Anti-parasitic methods and compositions utilizing diindolylmethane-related indoles

ABSTRACT

The present invention includes methods and compositions for the treatment and prevention of protozoal parasitic infections utilizing Diindolylmethane-related indoles. Additive and synergistic interaction of Diindolylmethane-related indoles with other known anti-parasitic and pro-apoptotic agents is believed to permit more effective therapy and prevention of protozoal parasitic infections. The methods and compositions described provide new treatment of protozoal parasitic diseases of mammals and birds including malaria, leishmaniasis, trypanosomiasis, trichomoniasis, neosporosis and coccidiosis.

1. FIELD OF THE INVENTION

The present invention relates to methods and compositions for thetreatment and prevention of parasitic infections, preferably protozoalparasitic infections, utilizing Diindolylmethane-related indoles.Administration of Diindolylmethane-related indoles, alone or incombination with known antiprotozoal agents, provides a method toselectively promote apoptosis of parasite infected host cells and ofextra-cellular protozoal parasites. Direct pro-apoptotic activity andinteraction of Diindolylmethane-related indoles with other knownanti-parasitic and pro-apoptotic agents is believed to permit moreeffective chemotherapy and to be complementary to co-administeredanti-protozoal vaccines. The methods and compositions of the presentinvention provide new therapeutic options in treating the majorprotozoal parasitic diseases of mammals and birds including malaria,leishmaniasis, trypanosomiasis, neosporosis and coccidiosis.

2. BACKGROUND OF THE INVENTION 2.1 Protozoal Related Disease

Protozoal parasites are single-celled organisms which live during someor all stages of their life cycle within organs, tissues and cells ofmulticellular, metazoan animals. As parasites, they obtain nutrientseither from the host organism's food supply or from its cells andtissues. As eukaryotic unicellular organisms, the protozoal parasitesare able to live both within animal cells and as free livingextra-cellular parasites residing in the blood, lymph tissue or withinthe intestinal lumen.

As agents of infection, the protozoal parasites are fundamentallydifferent than bacteria and viruses. Unlike bacteria and viruses,protozoa parasites are animals and share similar metabolism,respiration, and nutritional needs with their animal hosts. The similarmetabolism of protozoa to mammalian metabolism renders most antibioticsand antiviral agents, selectively active against bacteria and viruses,respectively, ineffective for protozoal infection. Activity of compoundswith antibacterial or antiviral activity against protozoal parasiteswould be atypical and unexpected. The lack of differences betweenprotozoal metabolism and host cellular metabolism requires novelpharmacologic approaches to find therapeutic agents selective foreliminating protozoal organisms living within an animal host. Unlikebacteria and viruses, protozoa may assume different sexual forms anddifferentiate into a variety of maturational stages in various organs,presenting unique challenges for recognition by the host immune system.As genetically more complex organisms than bacteria and viruses,protozoa differentiate into forms which resist killing by knownmicrobicides active against bacteria and viruses (Weir et al., 2002,Appl Environ Microbiol. 68(5):2576-9). Development of effectiveantiprotozoal therapeutics and stimulation of host immune responsesagainst protozoal parasites therefore requires approaches different fromthose utilized in developing antibacterial, antiviral, and generalimmune potentiating agents.

The primary protozoal parasites causing disease in man includehemoflagellates of the class Trypanosomatidea, causing Leishmaniasis andTrypanosomiasis, and parasites of the phylum Apicomplexa, classCoccidea, causing malaria, toxoplasmosis, cryptosporidiosis, andbebesiosis. Species of Coccidea can infect humans, domestic animals andlivestock, including poultry, lambs, calves, piglets, and rabbits.Protozoal parasitic diseases related to malaria include disease causedby parasites of the species Neospora. Neospora infections occur in dogs,cattle, sheep, goats and horses.

The majority of populations in developing countries are now at high riskof various protozoal infections including malaria, leishmaniasis, andtrypanosomiasis. Together these protozoal diseases cause millions ofpreventable deaths every year. No preventive or therapeutic vaccines areyet available for these parasitic diseases. The market for drugs againstsuch diseases is limited by poverty and the emergence of resistance toexisting single agent chemotherapy. As used herein, chemotherapy refersto the use of chemical substances to treat disease. Due to the lack ofprotective immunity following infection, with or without chemotherapy,reinfection is a common phenomenon. Innovative and cost effective newdrugs and combination therapies using new and old drug products areurgently needed. The development of broad-spectrum anti-parasitic agentsable to be used in combination with existing chemotherapeutics ispreferable to reduce the emergence of new resistance. An idealanti-protozoal drug would target multiple protozoan parasites, be activeby various routes of administration, reduce morbidity and mortalitycaused by such infections, not interfere with co-administered vaccinesas they become available, and reduce the need for hospital-basedtreatment.

The important protozoal sources of infection addressed by the methods oftreatment and compositions of the present invention are a subset ofprotozoal organisms within the biologic kingdom Protista. The protozoarelevant to the present invention are summarized in Table 1.

TABLE 1 Selected field of parasites relevant to treatment methods andcompositions using DIM-related indoles. Phylum Class Order Genus SpeciesChromista Microsporidia Parabasalea Trichomonadida Enterocytozoonbieneusi Parabasalia Bigyra Trypanosomatida Leishmania Blastocystishominis Euglenozoa Axostylata Adeleida Trypanosoma Trichomonas vaginalisSubphylum Kinetoplasta Eimeriida Cystoisospora Leishmania speciesApicomplexa (Sporozoa) Trypanosomatidea Haemosporida Plasmodiumdonovani, infantum (chagasi), Coccidea Conoidasida Eucoccidioridatropica, braziliensis, guyanensis Trypanosoma species Brucei, gambiense,rhodesiense Trypanosoma cruzi Trypanosoma rangeli Cryptosporidiumbaileyi Cryptosporidium meleagridis Cryptosporidium parvumCryptosporidium muris Cyclospora cayetanensis Isospora belliCystoisospora Toxoplasma gondii Plasmodium falciparum Plasmodiummalariae Plasmodium ovale Plasmodium vivax Babesia gibsoni Babesiamicroti Neospora Sarcosystis homins Suihominis, lindemanni

2.2 Scope of Protozoal Parasitic Infections

Parasitic protozoa are responsible for a variety of human diseasestransmitted by insect vectors, i.e., carriers, including malaria,leishmaniasis, and trypanosomiasis. Other protozoal parasites can betransmitted directly from other mammalian reservoirs or from person toperson. Lacking vaccines, vector control and selective chemotherapy havebeen the only ways to reduce transmission and treat infectedindividuals, respectively. Because the immune system plays a crucialrole in controlling protozoal infection, opportunistic infection withprotozoal organisms is an increasing problem in infants, cancerpatients, transplant recipients, and those co-infected with humanimmunodeficiency virus (HIV). Pregnancy also suppresses certain immunefunctions. New anti-protozal treatments are needed which are safer formother and fetus during pregnancy, particularly for malaria,toxoplasmosis, and trichomonas infections. Vaccines are needed whichovercome diminished immune responses and induce an adequate long termimmune response. Vaccines can be used in conjunction with compatiblechemotherapy to improve therapy of pre-existing chronic infection inendemic areas.

2.2.1 Malaria is an Uncontrolled Protozoal Disease

Malaria arises from infection with an Apicomplexan protozoan parasiteknown as Plasmodium. Only four species of the genus Plasmodium causehuman malaria. P. vivax is the most common and fatal. P. ovale and P.malariae are less common and have intermediate severity. P. falciparumis the most virulent, responsible for high infant mortality, andassociated with current drug resistance. The disease is transmitted tohuman beings through the bite of infected female Anopheles mosquitoesand by transfusion of infected blood.

Due to the emergence and spread of drug-resistant malaria parasites,pesticide-resistant malaria-transmitting mosquitoes, and populationgrowth in endemic areas, malaria now causes approximately 500 millionclinical cases per year. It is prevalent in children and pregnant women,causing about one million annual deaths in children under the age offive. Children growing up in rural and endemic areas are subject to morefrequent malaria related illness and deaths than more resistant adults.

The most severe form of Plasmodium falciparum infection is cerebralmalaria (CM). Cerebral malaria implies the presence of neurologicalfeatures, especially impaired consciousness. Treatment of CM is limitedto a few conventional anti-malarial drugs (quinine or artemisinins) andsupportive care including parenteral fluids, blood exchange transfusion,osmotic diuretics and correction of hypoglycemia, acidosis andhypovolemia. The management of CM includes prompt administration ofappropriate parenteral anti-malarial agents and early recognition andtreatment of the complications. In children, the complications includesevere anemia, seizures and raised intracranial pressure. In adults,renal failure and pulmonary edema are more common causes of death.

A number of drugs ranging from those of natural origin to synthetic oneshave been developed for the treatment of malaria. Quinine andartemisinin are the commonly known drugs of natural origin, which areused for the treatment of malaria. A number of synthetic anti-malarialdrugs such as chloroquine, mefloquine, primaquine, halofantrin,amodiaquine, proguanil, atovaquone, maloprim are known in theliterature. Quinidine Gluconate, Quinine Sulfate, typically incombination with Doxycycline hyclate, Clindamycin, orPyrimethamine-sufadoxine are also used for malaria. In chloroqineresistant strains, preferred oral therapy includes MefloquineHydrochloride and Atovaquone-proguanil hydrochloride combinations. Intreatment of infections with P. vivax, P. malariae, P. ovale, andchloroquine sensitive P. falciparum, chloroquine phosphate andprimaquine phosphate are used.

In recent years, drug resistant malaria has become one of the mostserious problems in malaria control. Drug resistance necessitates theuse of drugs which are more expensive and may have dangerous sideeffects. The emergence of resistance can be prevented by the use ofcombinations of drugs with different mechanisms of action. The use ofdrug combinations for all antimalarial treatment not only delays theonset of drug resistance, but also accelerates recovery and increasescure rates. A number of antimalarial combinations are already known inthe field of malarial chemotherapy. The specific combinations in use,dosages, and relative merits of various combinations have beensummarized (Kremsner et al., 2004, Lancet 364:285-94).

With the emergence of P. falciparum strains resistant to chloroquine andquinine, further alternative antimalarial chemotherapy is required. Dueto frequent re-infection following complete or partial treatment,vaccine therapy promoting long term immunity to re-infection is needed.New chemotherapy will preferably clear the current infection and notinterfere with co-administered vaccines as they become available.Preferred combinations of anti-malarials utilize drugs that overcomechloroquine resistance, have a good safety profile, and are welltolerated. Artemisinin, obtained from the plant Artemisia anua, and itsderivatives are rapidly effective in severe malaria. Artemisinincompounds have been evaluated in several centers and are found to beeffective, and safe (Miskra et al., 1995, Trans R Soc Trop Med Hyg89:299-301).

In addition, the patent literature describes the combination ofatovaquone and proguanil as a method for the treatment of malaria. SeeU.S. Pat. No. 5,998,449. The combination of fenozan with anotheranti-malarial agent selected from artemisinin, sodium artesunate,chloroquine, or mefloquine is described for the prophylactic andcurative treatment of malaria. See U.S. Pat. No. 5,834,505. Synergisticcombination kits using atemisinin derivatives, sulfadoxin andpyrimethamine for severe, multi-drug resistant malaria are described byTipathi et al. in U.S. Patent Application Publication No. 2006/0141024A1.

2.2.2 Trypanosomiasis Lacks Effective Chemotherapy for Early and LateDisease

African trypanosomiasis (sleeping sickness) is caused by a subspecies ofthe parasitic haemoflagellate, Trypanosoma brucei. The infection beginswith the bite of an infected tsetse fly (Glossina spp.). Two forms ofthe disease are known, one caused by Trypanosoma brucei rhodesiense,endemic in Eastern and Southern Africa, and the other caused by T. b.gambiense, originally detected in West Africa, but also widespread inCentral Africa. African Trypanosomiasis results in febrile,life-threatening illness in humans and also threatens livestock. T.brucei parasites rapidly invade the Central Nervous System (CNS) causingdeath within weeks if untreated. T. b. gambiense proliferates relativelyslowly and can take several years before infecting the CNS system. Thereare four important drugs approved to treat these infections. Two ofthese, pentamidine and suramin, are used before the CNS involvement. Thearsenic-based drug, melarsoprol is used in the case of infectionsestablished in the CNS. The fourth drug, eflornithine, is used againstlate stage infection caused by T. b. gambiense. This drug is ineffectiveagainst T. b. rhodesiense. Nifurtimox is another drug licensed for bothAmerican trypanosomiasis and melarsoprol-refractory late stage disease.

American trypanosomiasis or Chaga's disease is caused by Trypanosomacruzi and effects millions of people in South and Central America, andMexico. Untreated Chaga's disease causes decreased life expectancy dueto parasitic cardiomyopathy and heart failure, megaesophagus, andmegacolon. Blood-sucking triatomid bugs transmit the infection to youngchildren and transplacental infection can occur with parasitemia duringpregnancy. Nifurtimox and benznidazole are two drugs used for treatmentof the acute disease, but are not known to be therapeutic for thechronic infection in older children and adults. In the absence of aneffective vaccine, better agents are needed that can be takenprophylactically by at risk children. Following infection, additionalagents are needed to be used in conjunction with nifurtimox andbenznidazole to increase efficacy, permit lower doses of the currentagents with reduced toxicity, and shorten the currently requiredduration of treatment.

2.2.3 Leishmaniasis Lacks Practical and Safe Chemotherapy

Human leishmaniasis comprises a heterogeneous spectrum of diseases.Three major forms are generally distinguished: cutaneous leishmaniasis,mucocutaneous leishmaniasis and visceral leishmaniasis, of which thelatter is potentially lethal. They are caused by various species of theprotozoan parasite Leishmania and transmitted by female sandflies. Thedisease is currently estimated to affect some 12 million people in 88countries. Worldwide, leishmania/HIV co-infection is now considered anemerging disease where about 50% of adult visceral leishmaniasis casesare related to co-existing HIV infection.

The current treatment for leishmaniasis involves administration ofpentavalent antimony complexed to a carbohydrate in the form of sodiumstibogluconate (Pentosam or Sb(V)) or meglumine antimony (Glucantine),which are the only established anti-leishmanial chemotherapeutic agentswith a clearly favorable therapeutic index. The exact chemical structureand mode of action of pentavalent antimonials is still uncertain.Amphotericin B and Pentamidine are the second line of anti-leishmanialagents, but are reserved for non-responding infections due to potentialtoxicity. Since resistance to the antimony-based anti-Leishmanial drugsis emerging and treatment failures are common, new combination therapiesare needed. Miltefosine is a recently introduced oral drug effective forvisceral and cutaneous disease. The importance of this new oral agentextends to the treatment of dogs which serve as an important reservoirof the disease. The identification of additional, new and effectiveanti-leishmanial agents for oral administration would allow furthertreatment options, help prevent emerging resistance to Miltefosine andantimony-based drugs, and increase the chance for regional control ofleishmaniasis. DIM has been shown to be a potent inhibitor of Leishmaniadonovani topoisomerase I (LdTOPILS) with an IC₅₀ of 1.2 μM. See Roy A.,et al., Biochemical Journal, 8 Oct. 2007, Immediate PublicationManuscript BJ 20071286 (not the final version).

2.2.4 Trichomonal Disease

Trichomonal infection, typically vulvo-vaginitis in women and urethritisin men, is sexually acquired and one of the most common protozoalparasite infections in humans. In the United States, it is estimatedthat more than 2 million women are infected each year. Trichomonasvaginitis causes vulvar itching and an odorous vaginal discharge. It iscaused by Trichomonas vaginalis, a single-celled protozoan parasite notnormally found in the flora of the genitourinary tract. TypicallyTrichomonal infection is treated with oral metronidazole which is FDAapproved in various dosage regimens. Though efficacious, Metronidazolecan exhibit serious dose-related side effects, particularly on the bloodand on the central nervous system. Experiments show it to be mutagenicand carcinogenic. Recently, treatment failure and emerging resistance tometronidizole have been documented, indicating a need for moreconsistently effective therapies which will include combinations ofdrugs active against strains of T. vaginalis that may be resistant tometronidazole. Preferred treatments will include agents safe forpregnant women and allow lower doses of co-administered metronidazole.

2.2.5 Protozoal Disease in Immunocompromised Hosts

The risk of parasitic diseases is also present outside developingcountries and often takes the form of chronic diarrheal disease insubjects with underlying immune deficiency. These infections can becaused by Isospora belli, and Cyclospora cayetanensis, both coccidianprotozoa, where infection results in self-limited diarrhea in normalhosts and prolonged diarrhea in individuals with AIDS. Both infectionsrespond to treatment with timethroprim-sufamethoxazole. Cryptosporidiaare additional coccidian parasites that cause diarrhea in animal speciesand humans. Cryptosporidium parvum and C. Hominis account for mostcoccidial infections in humans. These organisms form oocytes, which whendigested release sporozoites that invade host epithelial cells,penetrating the cell membrane but not the enterocyte cytoplasm.Nitazoxanide is the only drug approved for the treatment ofcryptosporidiosis in the United States. The identification of additionaleffective anti-crytosporidial agents for oral use would allow additionaltreatment options for individuals with HIV infection who respondunpredictably to Nitazoxanide.

Toxoplasmosis, is a zoonotic infection by the obligate intracellularprotozoan, Toxoplasma gondii. Toxoplasmosis is found throughout theworld, including the United States. Cats and other feline species arethe natural hosts for Toxoplasma gondii, however tissue cysts(bradyzoites) have been recovered from all mammalian species examined.Pregnant women and those with weak immune systems are particularlysusceptible to the health risks resulting from Toxoplasma infection.Severe toxoplasmosis, particularly trans-placental exposure, can resultin damage to the brain, eyes, and other developing organs in utero.Currently available treatments for toxoplasmosis, which are the drugstrisulfa-pyrimdine, sulfadiazine and pyrimethamine, are not effective,and can be toxic to the host. Therefore, there is a need for therapeuticagents to treat toxoplasmosis that are more effective and less toxicthan currently available treatment agents. No available agent is used tocontrol Toxoplasmosis in cats.

2.3 Protozoal Cell Behavior Includes Apoptosis-Like Responses andSuppression of Apoptosis in Infected Host Cells

Apoptosis is the process of programmed cell death by which damaged cellsare eliminated upon generation of unopposed death signals within thedamaged cell. While apoptosis is primarily viewed as a biologic responseof multicellular organisms providing a means of eliminating infected ortransformed cells in the setting of viral and cancer-related disease,protozoal organisms have also been noted to exhibit programmed celldeath behavior (Lee et al., 2002, Cell Death Differ. 9:53-64). Wheninfecting host cells in mammals, protozoal parasites have also beennoted to suppress host cell apoptosis. For example, activation of theNuclear Factor Kappa B (NFκB) survival signaling pathway has beendescribed following infection by Trypanosoma cruzi (Petersen et al.,2006, Infect Immun. 74:1580-7).

2.4 Natural Indole Compounds can Influence Apoptosis

Cruciferous vegetables contain a family of plant protective compoundscalled glucosinolates which give rise to active compounds with indolerings exemplified by indole-3-carbinol (I3C). Oral ingestion of I3Cresults in the gastric conversion of I3C into at least twenty acidcondensation products, many of which are bioavailable, the mostprevalent of which include CTR (cyclic trimer;5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b′:7,8-b″]triindole), HI-IM(1-(3-hydroxymethyl)-indolyl-3-indolylmethane), DIM (diindolylmethane),ICZ (indolocarbazole) and LTr-1 (linear trimer;[2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane) (Stresser et al.,1995, Drug Metabolism and Disposition 23:965-975). The fact that thereare many non-DIM acid condensation products of I3C, produced in vivo atequal or greater levels as DIM, which can be responsible for I3C'sactivity, requires that biologic activities of individual condensationproducts like DIM be demonstrated directly.

As one of many products derived from I3C, DIM is also present incruciferous plants following release of I3C. Once formed, DIM is stablein acid. In cell culture, isolated DIM has been shown to have apoptosispromoting effects in both estrogen-dependent and independent breastcancer cells (Hong et al., 2002, Biochem Pharmacol. 63:1085-97). Inanimals, orally administered DIM inhibits the growth of certainchemically induced forms of breast cancer (Chen et al., 1998,Carcinogenesis 19:1631-9). Recently, DIM has been shown to specificallyinduce apoptosis in Human Papilloma Virus (HPV) oncogene alteredcervical cancer cell lines (Chen et al., 2001, J Nutr. 131:3294-3302).In further cell culture experiments, DIM has been shown to reduceactivation of the NFκB signaling pathway in breast cancer cells (Rahmanet al., 2005, Cancer Res. 65:364-71). Other non-DIM I3C condensationproducts were not tested. In vivo studies in mice suggest that expectedeffective plasma levels of DIM are not easily achieved in humans(Anderton et al., 2004, Drug Metab Dispos. 32:632-8).

In relation to its pro-apoptotic activity in tumor cells, DIM has alsobeen shown to be estrogenic in breast cancer cells (Riby et al., 2000,Biochem. Pharmacol. 60:167-177) and in rainbow trout, a model ofcarcinogenesis relevant to cancer in humans (Shilling et al., 2001,Toxicology and Applied Pharmacology 170:191-200). Since estrogeniceffects inhibit apoptosis, DIM may actually enhance estrogen relatedgrowth and survival of some cells. Based on the conflicting results ofDIM's activity in cell culture studies and estrogenic activity in vivo,it is difficult to predict DIM's effects in vivo on protozoal diseaseprocesses. In addition, DIM has been shown to activate the MitogenActivated Protein Kinase (MAPK) cell signaling pathway in cell culture(Leong et al., 2004, Mol Endocrinol. 18:291-302). Activated MAPK isassociated with cancer promotion, cancer cell survival, and inhibitionof apoptosis. These properties of DIM suggest that DIM would not beuseful as a promoter of apoptosis in protozoal infection.

One approach, that has not been developed for protozoal parasiticdisease, would be to selectively induce apoptosis in protozoal infectedcells and tissues in order to cause the programmed death of parasitesand of parasite infected cells. This would result in parasite clearanceand increased apoptosis may support the development of protective hostimmunity.

3. SUMMARY OF THE INVENTION

The present invention relates to Diindolylmethane (DIM) and DIM-relatedindoles that are useful for the treatment and prevention or reducing therisk of protozoal diseases in mammals and birds. The present inventionalso relates to compositions comprising DIM-related indoles, optionally,in combination with one or more additional antiprotozoal agents. Incertain embodiments, the compounds and methods of the present inventionare used for treatment or prevention or reducing the risk of infectionsby the primary protozoal parasites affecting humans, including, but notlimited to, hemoflagellates of the class Trypansomatidae, causingleishmaniasis and trypanosomiasis, flagellates of the class Axostylatacausing Trichomonal infection, and parasites of the phylum, Apicomplexa,causing malaria, toxoplasmosis, cryptosporidiosis, and bebesiosis. Alsocovered by the methods are treatment of infections by fungi of the genusCandida, particularly C. albicans, which behave like intracellularprotozoa by invading epithelial cells. In certain embodiments, thecompounds and methods of the present invention are used for treatment orprevention or reducing the risk of infections affecting non-humanmammals, including, but not limited to, protozoal parasitic diseasesfunctionally related to malaria caused by parasites of the speciesNeospora. Neospora infections are known to occur in dogs, cattle, sheep,goats and horses. In certain embodiments, the compounds and methods ofthe present invention are used for treatment or prevention or reducingthe risk of infections with species of Coccidea, also of the phylumApicomplexa, which infect humans, domestic animals and livestock,including poultry, lambs, calves, piglets, and rabbits. As used herein,a subject includes, but is not limited to, humans, domestic animals suchas dogs, and livestock such as poultry, sheep, lambs, piglets, rabbits,cattle, calves, goats and horses. In certain embodiments, the compoundsand methods of the invention are used for the treatment or prevention orreducing the risk of infection caused by Cyclospora, Isospora, orBlastocystis.

In certain embodiments, the methods of the invention employstructurally-related, synthetically-derived, substituteddiindolylmethane compounds referred to as DIM-related indoles to treatprotozoal parasite infections. In a particular embodiments, the one ormore DIM-related indoles of the invention are selected from the groupconsisting of 3,3′-diindolylmethane (DIM), hydroxylated DIMs,methoxylated DIMs, 2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR),hydroxylated LTRs, methoxylated LTRs, 5,5′-dimethylDIM (5-Me-DIM),2,2′-dimethylDIM (2-Me-DIM), 5,5′-dichloroDIM (5-Cl-DIM),imidazolyl-3,3′-diindolylmethane, nitro-substitutedimidazolyl-3,3′-diindolylmethanes,2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole,6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole,and 2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane, and indole-3carbinol (I3C).

In certain embodiments the DIM-related indole or indoles are used incombination with anti-protozoal therapeutics. Anti-protozoal agentswhich are combined with DIM-related indoles according to the presentinvention include established antiprotozoal drugs, natural productderived antiprotozoal drugs, apoptosis promoting drugs often with ahistory of use as apoptosis promoting chemotherapeutics, and naturalproducts from plants with antiprotozoal activity. Establishedantiprotozoal agents for combined use include atovaquone, amodiaquine,amphotericin, butoconazole, clindamycin, eflornithine, fumagillin,iodoquinol (diiodohydroxyquin), clioquinol (iodochlorhydroxyquin),Etanidazole, Benznidazole, fluoroquinolones, enoxacin, ciprofloxacin,doxycycline, melarsoprol, metronidazole, miltefosine, nifurtimox,nitazoxanide, paromomycin, pentamindine, sodium stibogluconate, suramin,tinidozole, pyrimethamine, proguanil (chloroguanide), spiramycin, andsulfadoxine. Natural product derived antiprotozoal drugs useful forcombined use include sesquiterpene lactones related to artemisinin fromArtemisia annua, particularly artemisinin, dihydroartemisinin,artemether, artesunate, and further derivatives of artemisinin,quinolines like quinine derived from the bark of the South Americanchinchona tree, including quinine and quinine-related quinolines,halofantrine, mefloquine, lumefantrine, amodiaquine, pyronaridine,piperaquine, chloroquine, hydryoxychloroquine, napthoquine, primaquine,and tafenoquine, curcuminoids derived from curcumin, an extract fromCurcuma domestica, including 6-gingerol and 6-paradol, coronaridine,18-methoxycoronaridine, selected flavonoids, including luteolin,extracts the fruit pericarp of Sapindus mukorossi, and extracts of Yuccaschidigera. Apoptosis promoting antiprotozoal agents for combined useinclude artemisinin derivatives, atovaquone, chloroquine, iodoquinol(diiodohydroxyquin), clioquinol (iodochlorhydroxyquin), sodiumstibogluconate, and curcumin. Some apoptosis promoting chemotherapeuticsused are also useful in combination with DIM-related indoles and includePyrroloquinazolinediamine, Novobiocin, cyclosporine, dihydrobetulinicacid, campothecins, especially topotecan, irinotecan, SN38 (the activemetabolite of irinotecan), bortezimib, etoposide, salvicine, anddoxorubicin. Antiprotozoal natural products useful with the methods ofthe present invention include teas and extracts made from Artemisiaannua, teas and extracts made from Curcuma domestica, extracts fromgarlic which include allicin and other thiosulfinates, root extracts ofUvaria chamae (Annonaceae) and Hippocratea Africana (Hippocrateaceae),and root extracts of Homalium letestui. Preferred agents for combineduse with DIM-related indoles include artemisinin extracts and relateddrugs, curcumin and curcumin-related drugs, and other antiprotozoalagents with short metabolic half lives. Specific, preferredantimalarials with rapid metabolism include artesunate,dihydroartemisinin, quinine, and clindamycin.

In a particular embodiment, the DIM-related indole and an antiprotozoalagent are administered simultaneously. In another embodiment, theDIM-related indole and antiprotozoal agent are administered within ashort time of one another, for example, 30 seconds, 1 minute, 5 minutes,15 minutes, 30 minutes, 1 hour, 4 hours, 8 hours, 12 hours or 24 hoursof one another.

In an additional embodiment, the combination of a DIM-related indole anda antiprotozoal agent is administered in conjunction with adifferentiation promoting agent which helps protozoa infected cellsdevelop into more completely differentiated and therapeuticallysensitive cells. Differentiation promoting agents include Vitamin-D,Vitamin-D derivatives, Vitamin-A (retinoids), 9-cis-Retinoic acid,13-cis-Retinoic acid, trans-Retinoic acid, all-trans-Retinol, retinylacetate, Retinyl palmitate, and granulcyte/macrophage colony stimulatingfactors including recombinant human Filgrastim and Sargramostim.

In an additional embodiment, the combination of a DIM-related indole anda antiprotozoal agent is administered in conjunction with anti-protozoalvaccines which contain attenuated protozoal organisms, typicallyinactivated by irradiation and/or chemical processing. Alternatively,the DIM-related indole, antiprotozoal agent, and anti-protozoal vaccine,are administered with an additional immune potentiating agent (which isnot a DIM-related indole). Immune potentiating agents useful in themethods of the present invention include aloe vera extracts, purifiedaloe mannans and acemanans, mushroom extracts, beta-glucans, andextracts of the root of North American ginseng (Panax quinquefolium)containing poly-furanosyl-pyranosyl-saccharides (CV Technologies Inc.,Edmonton). Beta-glucans include those derived from Saccharomycescerevisiae (ImmunDyne, Inc., Florence, Ky.). Other useful fungalextracts containing branched glucans are derived from mushrooms, such asthe maitake mushroom (Grifola frondosa).

The invention further provides compositions, for example, a compositioncomprising a therapeutically effective amount of the combination of DIM,or a DIM-related indole, and an anti-protozoal compound or combinationof anti-protozoal compounds. In particular embodiments, the compositionsare formulated for oral, sublingual, rectal, vaginal, parenteral, andtopical administration. In a further particular embodiment, thedifferent formulations are combined to form a kit combining rectalsuppositories with an oral suspension for pediatric use, or rectal orvaginal suppositories with capsules or tablets for adult use.

The present invention also provides compositions for treating protozoalinfections comprising DIM, a DIM-related indole, or DIM in combinationwith selected known antiprotozoal compounds, in an amount effective toreduce blood, tissue or intestinal parasite counts, formulated in theform of a dietary supplement, for example, a nutraceutical tablet,capsule, drink mix, or fortified food; a tea mix, or chewing gum.

In the methods and compositions of the invention, DIM or a DIM-relatedindole is preferably processed to increase bioavailablity and/ormicroencapsulated with phosphatidylcholine (PC), complexed with PC, ormade into rapidly dissolving microparticles and nanoparticles.Preferably, the formulations will include specialized vehicles forrectal and vaginal administration and be safe for use during pregnancy.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Diindolylmethane and DIM-relatedindoles that are useful for the treatment and prevention or reducing therisk of protozoal diseases in mammals and birds. In particular, theinvention relates to the treatment and prevention of the protozoaldiseases in mammals and birds in Table 1. In certain embodiments, aDIM-related indole is used in combination with one or more of thefollowing: an anti-protozoal agent, a differentiation agent, an immunepotentiating agent and an anti-protozoal vaccine. The present inventionis directed to compositions comprising a DIM-related indole, eitheralone or in combination with one or more additional antiprotozoalagents. The compounds and methods of the present invention may be usedfor treatment and control of infections by the primary protozoalparasites affecting man, including, but not limited to, hemoflagellatesof the family Trypansomatidae, causing Leishmaniasis, Trypanosomiasis,and Bebesiosis, flagellates of the class Axostylata causing Trichomonalinfection, and parasites of the Sporozoa phylum, Apicomplexa, causingmalaria, toxoplasmosis, and cryptosporidiosis. Also covered by themethods are treatment of infections by fungi of the genus Candida,particularly C. albicans, which behave like intracellular protozoa byinvading epithelial cells. In non-human mammals, protozoal parasiticdisease functionally related to malaria includes disease caused byparasites of the species Neospora. Neospora infections are known tooccur in dogs, cattle, sheep, goats and horses. Also treatable accordingto the methods and compositions of the present invention is infectionwith species of Coccidea, also of the phylum Apicomplexa, which infecthumans, domestic animals and livestock, including poultry, lambs,calves, piglets, and rabbits.

Without being bound by any theory, it is believed that the compounds andmethods of the present invention promote apoptosis, for example, by toinhibiting cell survival signaling in host cells where such signaling isa response protozoal parasite infection, which supports the induction ofhost immunity, especially when used in conjunction with anti-protozoalvaccines. Promotion of more efficient apoptosis and interaction ofprotozoal antigens with host immune cells is believed to result from thecombined use of DIM-related indole with a variety of anti-protozoalagents, especially anti-protozoal vaccines.

Protozoal infection of cells initiates cell-growth and cell-survivalmechanisms uniquely attributed to the interaction of protozoal parasitewith host cell apoptotic mechanisms (Heussler et al., 2001, Int JParasitol. 31:1166-76). Upon entry into cells, protozoal parasitesprovide an activation signal for cell survival including activation ofNFκB signalling which inhibits cellular apoptosis (Shapira, 2004, JParasitol. 34(3):393-400). In protozoal parasite infections, protozoalparasites utilize a variety of strategies to avoid interaction with thehost immune system including differentiation to less activating forms,inhibition of pro-apoptotic stress proteins, and inhibition of immuneactivating antigen display on the surface of host cell membranes.DIM-related indoles trigger pro-apoptotic signals through endoplasmicreticulum stress which has been shown to induce apoptosis in cancerouscells (Sun et al., 2004, Cell Stress Chaperones. 9(1):76-87).

Without being bound by any theory, the present invention employsDIM-related indoles alone and together with additional antiprotozoalagents to inhibit the protozoal parasite associated activation of NFκBand selectively induce apoptosis in parasite infected cells, therebyreducing production of protozoal progeny, reducing parasite load, andresolving or shortening the period of infection. Prophylactic uses ofDIM-related indoles alone or with antiprotozoal agents can preventprimary infection or re-infection with protozoal parasites. Selectiveinhibition of overactive survival and growth signals in protozoalparasite infected cells in the present invention can provide effectivetherapy, causing protozoa altered cells to be eliminated by triggeringprogrammed cell death (apoptosis). Importantly, the promotion ofapoptosis in protozoa infected cells can stimulate a more effectiveimmune response, enhancing natural immunity and improving the short andlong term benefit from co-administered anti-protozoal vaccines (James,2005, J Infect Dis. 191(10):1573-5).

In certain embodiments, these methods of the present invention employstructurally-related, synthetically-derived, substituteddiindolylmethane compounds administered orally, parenterally, vaginally,or per rectum. In a particular embodiment, a combination of DIM, or aDIM-related indole, and one or more known anti-protozoal agents areprovided. The methods and compositions provide improved treatment forprotozoal parasite infections.

The invention is based in part on expected additive and synergisticactivity in using particular combinations of DIM-related indoles andantiprotozoal agents to selectively promote apoptosis in protozoalinfected cells and apoptosis-like cell death in extracellular parasites.Combined use with DIM-related indole is believed to permit lower doseuse of antiprotozoal agent(s), reducing dose-related side effects ofthese drugs. In certain embodiments, the compositions of the inventioncan be used with differentiation promoting agents such as Vitamin-Dderivatives (calcitriol[1-alpha-25-dihydroxycholecalciferol]), retinoidderivatives (Vitamin-A, isotretinoin, retinoids), macrophage colonystimulating factors (Filgrastim and Sargramostim), and other immunepotentiating agents. The combination of a DIM-related indole andantiprotozoal agents is believed to induce promotion of apoptosisresulting in the selective elimination of protozoa infected cells, andcauses resolution of protozoal parasite related lesions of infectedtissues, particularly the intestines, liver, skin, heart, spleen, andblood.

4.1 Diindolylmethane-Related Indoles

The DIM-related indoles or DIM compounds useful in the methods andcompositions of the invention include DIM (3,3′-diindolylmethane) andthe related linear DIM trimer(2-(indol-3-ylmethyl)-3,3′-diindolylmethane [also written: 2(Indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane] (LTR), andIndole-3-Carbinol (I3C). As used herein, “DIM-related compound”,“DIM-related indole”, and “DIM derivative” are used interchangeably, andrefer to both natural metabolites and analogs of DIM, and also to“structurally-related, synthetically-derived, substituteddiindolylmethane compounds” and “synthetic derivatives of DIM”, such asthose disclosed herein and known in the art. As used herein,“cruciferous-related indoles” encompasses the terms “DIM-relatedcompound”, “DIM-related indole”, and “DIM derivative”. One of ordinaryskill in the art will recognize that in any of the pharmaceuticalcompositions or methods of the invention where DIM is used, aDIM-related compound, including a structurally-related,synthetically-derived, substituted diindolylmethane compound orsynthetic derivative of DIM, can be used.

The chemical structure of a DIM is as follows (where each of the Rgroups is H):

In particular embodiments, the DIM-related indole is a compound offormula R⁴², R⁵¹, R³⁵, R³⁶, R³⁷, R³⁸, R⁹⁰, R⁴¹, R⁵⁰, R³¹, R³², R³³, R³⁴and R⁹¹ individually and independently, are hydrogen or a substituentselected from the group consisting of a halogen, a hydroxyl, a nitro,—OR¹⁰⁰, —CN, —NR¹⁰⁰, R¹⁰¹, —NR¹⁰⁰, R¹⁰¹, R¹⁰²⁺, —COR¹⁰⁰, CF₃, —S(O)nR¹⁰⁰(n=0-2), —SO₂NR¹⁰⁰, R¹⁰¹, —CONR¹⁰⁰, R¹⁰¹, —NR¹⁰⁰COR¹⁰¹, —NR¹⁰⁰C(O)NR¹⁰¹,R¹⁰², —P(O)(OR¹⁰⁰)_(n) (n=1-2), optionally substituted alkyl, halovinyl,alkenyl, alkynyl, aryl, heteroalkyl, heteroaryl, or optionallysubstituted cycloalkyl or cycloakenyl, all of one to ten carbons andoptionally containing 1-3 heteroatoms O or N, wherein R¹⁰⁰, R¹⁰¹ andR¹⁰² are optionally substituted alkyl, alkenyl, alkynl, aryl,heteroalkyl, heteroaryl of one to ten carbons, and R⁹⁰ and R⁹¹ mayfurther be O to create a ketone. In particular embodiments, the compoundincludes at least one such substituent, preferably at a position otherthan, or in addition to R⁴² and R⁴¹, the linear or branched alkyl oralkoxy group is one to five carbons, and/or the halogen is selected fromthe group consisting of chlorine, iodine, bromine and fluorine.

In certain embodiments, an active hydroxylated or methyoxylatedmetabolite of DIM, i.e., a compound of formula I, wherein R³², R³³, R³⁶,and R³⁷ are substituents independently selected from the groupconsisting of hydrogen, hydroxyl, and methoxy, and R³¹, R³⁴, R³⁵, R³⁸,R⁴¹, R⁴², R⁵⁰, and R⁵¹ are hydrogen, is utilized. In particularembodiments, the DIM-related indole is a mono- or di-hydroxylated DIMderivatives at carbon positions 2, 4-7 and 2′, and 4′-7′, including eachof [2, 4, 5, 6 or 7]-monohydroxy-DIM or [2′, 4′, 5′, 6′ or7′]-monohydroxy-DIM (e.g. 2-hydroxy-DIM, 4-hydroxy-DIM, etc.); and eachof [2, 4, 5, 6 or 7], [2, 4, 5, 6 or 7]-dihydroxy-DIM, [2′, 4′, 5′, 6′or 7′], [2′, 4′, 5′, 6′ or 7′]-dihydroxy-DIM, or [2, 4, 5, 6 or 7], [2′,4′, 5′, 6′ or 7′]-dihydroxy-DIM (e.g. 2,4-dihydroxy-DIM,2,5-dihydroxy-DIM etc, 2,2′-dihydroxy-DIM, 2,4′-dihydroxy-DIM etc.);particularly bilaterally symmetrical species, such as2,2′-dihydroxy-DIM.

In particular embodiments, the indolyl moieties are symmetricallysubstituted, wherein each moiety is similarly mono-, di-, tri-, para-,etc. substituted. In other particular embodiments, R⁴², R⁵¹, R³⁵, R³⁷,R³⁸, R⁹⁰, R⁴¹, R⁵⁰, R³¹, R³³, R³⁴ and R⁹¹ are hydrogen, and R³⁶ and R³²are a halogen selected from the group consisting of chlorine, iodine,bromine and fluorine. Representative compounds include, but are notlimited to, 3,3′-diindolylmethane, 5,5′-dichloro-diindolylmethane;5,5′-dibromo-diindolylmethane; and 5,5′-difluoro-diindolylmethane.Additional preferred such DIM derivatives include compounds wherein R⁴²,R⁵¹, R³⁵, R³⁷, R³⁸, R⁹⁰, R⁴¹, R⁵⁰, R³¹, R³³, R³⁴ and R⁹¹ are hydrogen,and R³⁶ and R³² are an alkyl or alkoxyl having from one to ten carbons,and most preferably one to five carbons. Representative compoundsinclude, but are not limited to, 5,5′-dimethyl-diindolylmethane,5,5′-diethyl-diindolylmethane, 5,5′-dipropyl-diindolylmethane,5,5′-dibutyl-diindolylmethane, 5,5′-dipentyl-diindolylmethane,5,5′-dimethoxy-diindolylmethane, 5,5′-diethoxy-diindolylmethane,5,5′-dipropyloxy-diindolylmethane, 5,5′-dibutyloxy-diindolylmethane, and5,5′-diamyloxy-diindolylmethane.

Additional preferred DIM derivatives include compounds wherein R⁵¹, R³⁵,R³⁶, R³⁷, R³⁸, R⁹⁰, R⁵⁰, R³¹, R³², R³³, R³⁴ and R⁹¹ are hydrogen, andR⁴² and R⁴¹ are an alkyl or alkoxyl having from one to ten carbons, andmost preferably one to five carbons. Representative compounds include,but are not limited to, N,N′-dimethyl-diindolylmethane,N,N′-diethyl-diindolylmethane, N,N′-dipropyl-diindolylmethane,N,N′-dibutyl-diindolylmethane, and N,N′-dipentyl-diindolylmethane. Inyet another embodiment, R⁴², R³⁵, R³⁶, R³⁷, R³⁸, R⁹⁰, R⁴¹, R³¹, R³²,R³³, R³⁴ and R⁹¹ are hydrogen, and R⁵¹ and R⁵⁰ are alkyl of one to tencarbons, and most preferably one to five carbons. Representativecompounds include, but are not limited to,2,2′-dimethyl-diindolylmethane, 2,2′-diethyl-diindolylmethane,2,2′-dipropyl-diindolylmethane, 2,2′-dibutyl-diindolylmethane, and2,2′-dipentyl-diindolylmethane. In another embodiment, R⁴², R⁵¹, R³⁵,R³⁷, R³⁸, R⁹⁰, R⁴¹, R⁵⁰, R³¹, R³³, R³⁴ and R⁹¹ are hydrogen, and R³⁶ andR³² are nitro.

In an alternative embodiment, active DIM derivatives with R₃₂ and R₃₆substituents made up of ethoxycarbonyl groups, and R₅₀, R₅₁ are eitherhydrogen or methyl, are utilized.

In another embodiment, active substituted DIM derivatives includingmethylated and chlorinated compounds, exemplified by those that include5,5′-dimethylDIM (5-Me-DIM), 2,2′-dimethylDIM (2-Me-DIM), and5,5′-dichloroDIM (5-Cl-DIM) are described in U.S. Patent ApplicationPublication No. 20020115708 by Safe, published Aug. 22, 2002,incorporated herein by reference in its entirety, are utilized in thepresent invention. In another embodiment, active DIM derivatives includeimidazolelyl-3,3′-diindolylmethane, including nitro substitutedimidazolelyl-3,3′-diindolylmethanes, and additional DIM-relatedcompounds described in U.S. Patent Application Publication No.2004/0043965 by Jong, Ling, published Mar. 4, 2004, incorporated hereinby reference in its entirety, are utilized. In a further embodiment,active DIM derivatives described in U.S. Pat. No. 6,656,963, U.S. Pat.No. 6,369,095 and U.S. Patent Application Publication No. 20060229355 byBjeldanes et al., published Oct. 12, 2006, incorporated herein byreference in its entirety, are utilized.

The chemical structure of LTR is as follows (where each of the R groupsis H):

In certain embodiments, an active hydroxylated or methyoxylatedmetabolite of LTR, i.e., a compound of formula II, wherein R⁶², R⁶³,R⁶⁶, R⁶⁷, R⁷⁰, and R₇, are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, and R⁶¹, R⁶⁴, R⁶⁵,R⁶⁸, R⁶⁹, R⁷², R⁸¹, R⁸², and R⁸³ are hydrogen, is utilized.

In certain embodiments, a DIM related compound has formula (III):

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₆-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms; and

R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl, with the provisos that: at leastone of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is otherthan hydrogen; and when R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are selectedfrom hydrogen, halo, alkyl and alkoxy, then R¹¹ and R¹² are other thanhydrogen and alkyl.

A preferred embodiment includes the use of2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole(SRI13668 (SRI Inc., Menlo Park, Calif.)). Additional preferredembodiments include the use of6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole(SRI Inc., Menlo Park, Calif.).

In another embodiment, a DIM related compound has formula (IV):

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are substituents independentlyselected from the group consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄ alkoxycarbonyl, C₆-C₂₀aryloxycarbonyl, halocarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, mono-(C₁-C₂₄alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substituted carbamoyl,mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido, cyano,isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, with the proviso that one butnot both of R² and R⁶ is amino, mono-substituted amino, ordi-substituted amino;

R¹¹ and R¹² are independently selected from the group consisting ofhydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl;

R¹³ and R¹⁴ are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ withthe proviso that at least one of R¹³ and R¹⁴ is other than hydrogen; and

X is O, S, arylene, heteroarylene, CR¹⁵, R¹⁶ or NR¹⁷ wherein R¹⁵ and R¹⁶are hydrogen, C₁-C₆ alkyl, or together form ═CR¹⁸, R¹⁹ where R¹⁸ and R¹⁹are hydrogen or C₁-C₆ alkyl, and R¹⁷ is as defined for R¹¹ and R¹².

A preferred embodiment includes the use of2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane (SRI Inc., MenloPark, Calif.).

In another embodiment, a DIM related compounds has formula (V):

wherein:

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², and X are defined as forcompounds of formula (III); and

R²⁰ and R²¹ are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸.

In yet another embodiment, the DIM-related indole is anindole-3-carbinol tetrameric derivative (Brandi et al., 2003, CancerRes. 63:4028-4036). In a further embodiment the DIM-related indole is anindole-3-carbinol derivative described as an anti-tumor agent (Weng J R,Tsai C H, Kulp S K, Wang D, Lin C H, Yang H C, Ma Y, Sargeant A, Chiu CF, Tsai M H, Chen C S. A potent indole-3-carbinol derived antitumoragent with pleiotropic effects on multiple signaling pathways inprostate cancer cells. Cancer Res. 2007 Aug. 15; 67(16):7815-24).

Substituted DIM analogs are readily prepared by condensation offormaldehyde with commercially available substituted indoles. Precursorcompounds can be synthesized by dimethylformamide condensation of asuitable substituted indole to form a substituted indole-3-aldehyde.Suitable substituted indoles include indoles having substituents at R⁴²,R⁵¹, R³⁵, R³⁶, R³⁷ and R³⁸ positions. These include, but are not limitedto 5-methoxy, 5-chloro, 5-bromo, 5-fluoro, 5′-methyl, 5-nitro, n-methyland 2-methyl indoles. The substituted indole 3-aldehyde product istreated with a suitable alcohol such as methanol and solid sodiumborohydride to reduce the aldehyde moiety to give substituted I3Cs.Substituted DIMs are prepared by condensing the substitutedindole-3-carbinol products. This may be achieved, for example, bytreatment with a phosphate buffer having a pH of around 5.5-7.4.

4.2 Combination Therapy

In certain embodiments of the invention, a DIM-related indole may beused in combination with one or more of the following: an anti-protozoalagent, a differentiation agent, an immune potentiating agent or ananti-protozoal vaccine. It is understood that more than one agent fromeach class can be used, for example, multiple anti-protozoal agents.

Particularly preferred drug combinations to be used in conjunction withadditional DIM-related indoles according to the present inventioninclude the non-artemisinin combinations quinine andsulfadoxine-pyrimethamine and quinine and doxycycline. Artemisinin basedcombination treatments to be used with DIM-related indoles includeArtemether-lumefantrine, artesunate and amodiaquine, dihydroartemisininand piperaquine, artesunate and mefloquine, artesunate andsulfadoxine-pyrimethamine, anddihydroartemisinin-napthoquine-trimetoprim. For crytosporidialinfections DIM-related indole, artemisinin and curcumin or genistein area preferred combinations. For Trichomonal infections a DIM-relatedindole is used with artesunate, metronidazole, tinidazole, orhexadecylphosphocholine (miltefosine). For Candidal infections aDIM-related indole is used with an azole derivative, particularly animidazole compound like clotrimazole, miconazole, or butoconazole. Forcombined bacterial and Trichomonal vaginitis, a DIM-related indole isused in combination with clindamycin, or with both clindamycin andartesunate. Alternatively, multi-organism infection can be treated witha DIM-related indole combined with metronidazole, or with bothmetronidazole and artesunate.

4.2.1 Anti-Protozoal Agents

Anti-protozoal agents which can be combined with DIM-related indolesaccording to the present invention include established antiprotozoaldrugs, natural product derived antiprotozoal drugs, apoptosis promotingdrugs often with a history of use as apoptosis-promotingchemotherapeutics, and known antiprotozoal natural products. As usedherein, an “anti-protozoal agent” does not include a DIM-related indole(which has anti-protozoal activity). In other words, in referring to thecombination of a DIM-related indole and an anti-protozoal agent, the“anti-protozoal agent” is an agent other than a DIM-related indole.

Antiprotozoal agents include, but are not limited, atovaquone;diaminopyrimidines, especially amodiaquine, amphotericin, butoconazole,astemizole clindamycin, eflornithine, fumagillin; the8-hydroxyquinolines, iodoquinol (diiodohydroxyquin), clioquinol(iodochlorhydroxyquin), the 2-nitroimidazoles, Etanidazole, Benznidazolefluoroquinolones, enoxacin, ciprofloxacin; doxycycline, melarsoprol,metronidazole, tinidazole, miltefosine, nifurtimox, nitazoxanide,paromomycin, pentamindine, sodium stibogluconate, antimony gluconate(SAG), and related antimonials, suramin, including the sodium salt,tinidazole, pyrimethamine, proguanil (chloroguanide), spiramycin, andsulfadoxine. Also useful are detergent and non-detergent spermacidesthat have additional anti-protozoal activity when used in topicalformulations (Gupta G. Microbicidal spermicide or spermicidalmicrobicide? Eur J Contracept Reprod Health Care. 2005 December;10(4):212-8).

Natural product derived antiprotozoal drugs include, but are not limitedto, sesquiterpene lactones related to artemisinin from Artemisia annua,particularly artemisinin, dihydroartemisinin, artemether, artesunate,and further derivatives of artemisinin described in the literature(Haynes, 2006, From artemisinin to new artemisinin antimalarials:biosynthesis, extraction, old and new derivatives, stereochemistry andmedicinal chemistry requirements. Curr Top Med. Chem. 6(5):509-37);quinolines like quinine derived from the bark of the South Americanchinchona tree, including alkaloids structurally related to quinine,quinine and quine-related quinolines, halofantrine, mefloquine,lumefantrine, amodiaquine, pyronaridine, piperaquine, chloroquine,hydryoxychloroquine, napthoquine, primaquine, tafenoquine, amodiaquineand 4-aminoquinolines derived from the quinolines (Neill et al., 2006,Curr Top Med Chem. 6:479-507); other quinones including those extractedfrom Salvia prionitis, particularly salvicine and its derivatives (QingC. et al, In vitro cytotoxicity of a salvicine, a novel diterpenoidquinone, Zhongguo Yao Li Xue Bao. 1999 April; 20(4):297-302);curcuminoids derived from curcumin, extracted from Curcuma domestica,including 6-gingerol and 6-paradol (Surh et al., 1999, J Environ PatholToxicol Oncol. 18:131-9); selected flavonoids and isoflavones,including, but not limited to, Genistein from soy, and derivatives froma number of plant sources including dehydrosilybin, silybin A andsilybin B and isosilybin A and isosilybin B, and 8-(1;1)-DMA-kaempferide (Tasdemir et al., 2006, Antimicrob Agents Chemother.50:1352-64), luteolin, baicalein, dihydrobetulinic acid, quercetin,eriodictyol acid, lursolic acid, oleanolic acid; and triterpenes,particularly Ganoderic acid X, isolated from Ganoderma amboinense andtriterpene rich extracts of Sapindus mukorossi known to haveanti-trichomonal activity.

Apoptosis promoting antiprotozoal agents include, but are not limitedto, artemisinin derivatives, atovaquone, chloroquine, iodoquinol(diiodohydroxyquin), clioquinol (iodochlorhydroxyquin), Jasmonic acid[3-oxo-2-(2-pentenyl)cyclopentaneacetic acid], methyl jasmonate[methyl3-oxo-2-(2-pentenyl)cyclopentaneacetic acid], andcis-jasmone[3-methyl-2-(2-pentenyl)-2-cyclopenten-1-one],3,3′-dihexyloxacarbocyanine iodide, sodium stibogluconate, extracts ofYucca schidigera, and curcumin. Apoptosis promoting chemotherapeuticsinclude, but are not limited to, Pyrroloquinazolinediamine, Novobiocin,quercetin, cyclosporine, dihydrobetulinic acid, campothectins,especially topotecan, irinotecan, SN38 (the active metabolite ofirinotecan), bortezimib, etoposide, quinones including salvicine, andanthracyclines including doxorubicin, daunorubicin, 4′-epirubicin,idarlibicin, and deoxydoxorubicin.

Antiprotozoal natural products include, but are not limited to, teas andextracts made from Artemisia annua, teas and extracts made from Curcumadomestica, extracts from garlic which include allicin and otherthiosulfinates, root extracts of Uvaria chamae (Annonaceae) andHippocratea Africana (Hippocrateaceae), and root extracts of Homaliumletestui. Also useful are extracts of Brasilian plants with demonstratedanti-malarial activity, including Vernonia brasiliana and Acanthospermumaustrale (Carvalho, 1991, Braz J Med Biol Res. 24(11):1113-23, andBotsaris AS. Plants used traditionally to treat malaria in Brazil: thearchives of Flora Medicinal. J Ethnobiol Ethnomed. 2007 May 1; 3:18).

Preferred agents for combined use with DIM-related indoles includeartemisinin extracts and related drugs, curcumin and curcumin-relateddrugs, and other antiprotozoal agents with short metabolic half lives,preferably with a half-life similar to DIM (i.e., approximately 2-7hours). In use with DIM-related indoles preferred antimalarials will bemetabolically cleared within 12 hours following an oral dose and notincrease activity of drug metabolizing cytochrome enzymes (Giao et al.,2001, Clin Pharmacokinet. 40(5):343-73). Additional useful naturalproduct preparations are described in the literature (Willcox et al.,2004, BMJ. 329(7475):1156-9).

4.2.2 Differentiation Promoting Agents

In an additional embodiment, the combination of a DIM-related indole anda known antiprotozoal agent is administered in conjunction withdifferentiation promoting agents which help protozoa infected cellsdevelop into more completely differentiated and more therapeuticallysensitive cells. Differentiation promoting agents include Vitamin-D,Vitamin-D derivatives, Vitamin-A (retinoids), retinoid derivatives, andgranulocyte/macrophage colony stimulating factors including recombinanthuman Filgrastim and Sargramostim.

4.2.3 Immune Potentiating Agents

The DIM-related indoles can also be used in combination with non-DIMrelated indole immune potentiating agents. Immune potentiating agentsuseful in the methods of the present invention include Aloe veraextracts, mushroom extracts, beta-glucans, and extracts of the root ofNorth American ginseng (Panax quinquefolium) containingpoly-furanosyl-pyranosyl-saccharides (CV Technologies Inc., Edmonton).Useful Panax quinquefolium extracts are described in U.S. Pat. No.6,083,932 by Pang et al. which is herein incorporated by reference inits entirety. Beta-glucans include those derived from Saccharomycescerevisiae (En-Bio Technology Co., Ltd.). Other useful fungal extractscontaining branched glucans are derived from mushrooms, such as themaitake mushroom (Grifola frondosa). Oral use of beta-glucans ininfections has been described (Jung et al., 2004, J Vet Med B Infect DisVet Public Health. 51(2):72-6).

4.2.4 Anti-Protozoal Vaccines

Existing vaccines do not induce sufficient protective immunity to clearexisting infection and prevent a repeat infection. Therefore, in oneaspect of the invention, a DIM related indole is administered with avaccine to improve the response to the vaccine, i.e., by inducing agreater response through inducing apoptosis.

In one embodiment, a DIM-related indole, optionally with anantiprotozoal agent, is administered in conjunction with ananti-protozoal vaccine, for example, a vaccine which contains attenuatedprotozoal organisms, typically inactivated by irradiation and/orchemical processing. Promotion of more efficient apoptosis of host cellsinfected with attenuated protozoa and more effective interaction ofprotozoal antigens with host immune cells is believed to result from thecombined use of DIM-related indole with a variety of vaccines.

4.3 Prevention and Treatment of Protozoal Parasite Infections.

In certain embodiments, the present invention provides for theprevention and treatment of protozoal parasite infections which includeactive infections and asymptomatic chronic infections. Currently, thisspectrum of protozoal parasite infections lacks adequate and effectivetherapy due to failure of previous approaches to selectively eliminateprotozoal parasite infected cells and tissues and to target theanti-apoptotic cell signaling pathways activated by protozoal parasites.Ideally, use of DIM-related indoles will allow safer and more effectivetreatments of protozoal infections during pregnancy in immunecompromised patients. Treatment of the various protozoal parasiticdiseases varies according to the parasite type and setting fortreatment.

4.3.1 General Principles of Treatment for Protozoal Parasites UsingDIM-Related Indoles

DIM-related indoles are used for the treatment and prevention ofprotozoal parasite infections in formulations most suitable for thedisease entity, disease severity, and setting of treatment. An objectiveof the invention is to eliminate or shorten the need for parenteraltherapy of protozoal diseases and, in doing so, reduce the need forhospitalization. When appropriate DIM is used in combination with otherdesirable antiprotozoal agents to reduce the chance of selecting forresistance to DIM-related indole activity. Desirable antiprotozoalagents for combined use possess one or more of the following importantcharacteristics: safety, stability, chemical compatability informulations, tolerability, effectiveness, oral or intravenous dosageform, short elimination half-time, independent mode of action,synergistic activity in vivo. Ideally, agents for combination will havelow cost and allow a short duration of use in order to achieve durabletreatment response, and be appropriate for both adult and pediatric use.

Also important in combined uses and products is compatibility with usein conjunction with antiprotozoal vaccines, including those which may bedeveloped in the future. Desirable antiprotozoal agents for combined useinclude compounds which like DIM-related indoles support selectiveapoptosis of parasite infected cells. Examples include, but are notlimited to, artemisinin derivatives, atovaquone, chloroquine, iodoquinol(diiodohydroxyquin), clioquinol (iodochlorhydroxyquin), miltefosine,sodium stibogluconate, and curcumin.

Also desirable for use in combination with DIM-related indoles, with orwithout vaccines, are selected antibiotic or chemotherapeutic agentswhich promote apoptosis. Desirable agents inhibit proteosomal function,induce endoplasmic reticulum stress, inhibit topoisomerase enzymes,inhibit vacuolar-H⁺-ATPase, and/or inhibit farnesyl transferase enzymes.Preferred chemotherapeutic agents for use in combination withDIM-related indoles for protozoal parasites are fluoroquinolones,enoxacin, ciprofloxacin, Novobiocin, cyclosporine, luteolin,butoconazole, sodium butyrate (Sodium butanoate), phenylbutyrate,curcumin, evodiamine, dihydrobetulinic acid, campothecins, especiallytopotecan, irinotecan, bortezimib, etoposide, quinones includingsalvicine, and the anthracyclines including doxorubicin, daunorubicin,4′-epirubicin, idarubicin, and deoxydoxorubicin.

Desirable antiprotozoal agents for combination with DIM-related indolescan also be chosen to be appropriate for out-of-hospital, rural usesettings. Such agents are intended for distribution and use asnutraceutical products without prescription or medical supervision. Theingredients need to be safe, stable without refrigeration, and simple touse. Ideally, nutraceutical products will be compatible for use alongwith other natural product antiprotozoals, especially tea brewed fromAtemesia annua, the source of artemisinin. Evodiamine, an indolealkaloid component extracted from the fruit of Evodiae Fuctus (Evodiarutaecarpa Benth.) is an alternative natural product for use withDIM-related indoles. Additional, DIM-related indole compatible herbalmedicines and appropriate use is described in the literature (Willcox etal., 2004, BMJ 329(7475):1156-9).

Dosages and treatment regimens for antimalarial agents are taught inGoodman & Gilman's The Pharmacologic Basis of Therapeutics, Goodman etal. [ed], 9th edition (Jan. 15, 1996) McGraw Hill Text.

4.3.2 Use of DIM-Related Indoles with Antiprotozoal Agents for Malaria

4.3.2.1 Severe Malaria

In severe Plasmodium falciparum infection with neurological features,termed cerebral malaria (CM), especially impaired consciousness, use ofDIM-related indoles is by parenteral route or rectal suppositories.Intravenous DIM-related indole is generally administered in rotationwith parenteral quinine or an artemisinin-based drug (e.g.,dihydroartemisinin, artemether, or artesunate). The rate of intravenousadministration of DIM-related indole and additional anti-malarialagent(s) is based on renal function, hepatic function and overallcondition of the patient. This is in addition to parenteral fluids,blood exchange transfusion, osmotic diuretics and correction ofhypoglycemia, acidosis and hypovolemia. Oxygen by mask or mechanicalventilation is added to potentiate the anti-parasitic activity of theDIM-related indole and additional anti-protozoal drug. Administration ofoxygen therapy is utilized to support the apoptotic mechanisms of actionof DIM-related indoles.

4.3.2.2 Moderate-Severe Malaria

Moderately severe malaria is typically characterized by fever andlethargy. When possible, blood tests for parisitemia should be performedto establish the diagnosis and be repeated to document the parasiteclearance time (PCT). According to the present invention, treatment isbegun with rectal DIM-related indole, preferably in a combinedsuppository with Artemisinin derivative. Those patients able to takeoral fluids simultaneously begin oral DIM-related indole preparation asa suspension or in capsules or tablets. Those subjects with diminishedlevel of consciousness are provided parenteral hydration fluids. Whenavailable, oxygen by mask is added to potentiate the anti-parasiticactivity of the DIM-related indole and additional anti-protozoal drug.Repeat blood smear examination is performed after 3 days to establishPCT. Oral DIM-related indole therapy is generally given with a secondantimalarial drug, preferably an artemisinin or artemisinin derivative,in combination with lumefantrine, mefloquine, sulfadoxine-pyrimethamine,or amodiaquine in chloriquine resistant areas. For use in chloroquinesensitive areas, DIM-related indole therapy is generally given withartimisinin and/or chloroquine. Optimal treatment duration is from 3 to14 days. A repeat blood smear for parisitemia is again performed at 14days and again at 28 days.

4.3.2.3 Use with Anti-Malaria Vaccines

Following the administration of a preventive malaria vaccine, typicallya sporozoite-based vaccine, oral DIM-related indole therapy is initiated24-96 hrs following receipt of the vaccine. Optimally, the DIM-relatedindole composition is given as an oral suspension or tablet incombination with one or more antimalarial drugs selected fromartemisinin derivatives, primaquine, atovaquone, proguanil, orpryimentamine-sulfadoxine.

4.3.2.4 Prophylactic Use by Non-Immune Individuals.

In anticipation of exposure to malarial parasites during travel orresisdence in an endemic area, DIM-related indole therapy is initiatedon arrival, continued daily during exposure to risk and furthercontinued 1-2 weeks after the period of exposure. Alternatively, theDIM-related therapy is taken in combination with Chloroquine phosphate(Aralen) 500 mg taken once weekly or in suggested pediatric dosesbeginning one week prior to exposure and continuing until 2 weeks afterexposure in chloroquine sensitive areas. Alternatively, in chloroquineresistant areas, the oral DIM-related indole therapy is taken inconjunction with Atovaqone-proguanin (Malarone) in standard doses, withMefloquine hydrocholorid (Lariam) in standard doses, with Doxycyclinehyclate in standard doses, or with Primaquine phosphate in standarddoses. At the option of a supervising physician, the dose of the non-DIMagents can be reduced during the period of prophylaxis when usingDIM-related indole therapy. Preferably, the DIM-related indole iscombined in the same tablet or capsule with primaquine phosphate.DIM-related indole is combined with an Artemisinin derivative, andoptionally additional curcumin for use during pregnancy.

4.3.3 Combined Use of DIM-Related Indoles with Antiprotozoal Agents forTrypanosomiasis

In treating advanced African trypanosomiasis during the latemenigoencephalitic stage an intravenous suspension or emulsion ofDIM-related indole is alternated with intravenous melarsoprol. Thecombined use is intended to allow a reduction of the melarsoprol dosefrom the typical 2.0-3.6 mg/kg per day dose of melarsoprol given for 3-4days once weekly for 3 consecutive weeks. Alternatively, lower doses ofmelarsoprol from 1-2 mg/kg per day are given with intravenousDIM-related indole for 10 continuous days. For infection due to T.brucei gambiense, intravenous or oral DIM-related indole is used inconjunction with intramuscular or intravenous Pentamidine at standarddoses for a period of 10 days. For infection due to T. bruceirhodensiense, intravenous or oral DIM-related indole is used inconjunction with intravenous Suranim at standard doses for a period of10 days. Alternatively, in severe cases of African trypanosomiasis,intravenous DIM-related indole is administered in conjunction withintravenous campothecin derivative such as topotectan in standard dosesfor 10 or more days.

In treating American trypanosomiasis (Chaga's Diseasae) due to T. Cruzi,intravenous or oral DIM-related indole is used in conjunction withintravenous Suranim at standard doses for a period of 10 days for severedisease. Alternatively, oral DIM-related indole is administered inconjunction with oral nifurtimox and/or oral bensnidazole.Alternatively, DIM-related indole is formulated with nifurtimox orbensnidazole in the same flavored oral suspension or pill to improvecompliance and ease of use during chronic treatment.

Alternatively, intravenous DIM-related indole is administered inconjunction with intravenous campothectin derivative such as topotectanin standard doses for 10 or more days in severe cases of Americantrypanosomiasis.

4.3.4 Combined Use of DIM-Related Indoles with Antiprotozoal Agents forLeishmaniasis

For the treatment of Leishmaniasis, DIM-related indole therapy iscombined with administration of Pentavalent antimony compounds for amore effective therapy. Typically oral or intravenous DIM-related indoleis administered in conjunction with sodium stibogluconate given at adose of 20 mg/kg/day or less for a period of 28 days to treat visceralleishmaniasis. Alternatively, intravenous DIM-related indole isadministered in conjunction with intravenous campothectin derivativesuch as topotectan in standard doses for 10 or more days in severe casesof visceral leishmaniasis. For cutaneous disease the duration ofcombined treatment is reduced to 20 days. Optionally, a transdermalpreparation of DIM-related indoles is added to the orally administeredDIM-related indole and applied topically in cutaneous disease.Alternatively, intravenous DIM related indole is administered incombination with liposomal amphotericin B where resistance toantimony-based drugs is identified. When possible, oral therapy forcutaneous disease consists of DIM-related indole given with oralmiltefosine. This combination permits a reduction in the standard doseof miltefosine from 100 mg/kg/day. In addition, a DIM-related indole canbe administered both orally and topically for use in combination withparomomysin and/or additional gentamicin.

In a alternative preventive approach, a DIM-related indole isadministered orally or parenterally with miltefosine and/or aartimesinin derivative in a veterinary formulation formulated as asupplement for dogs or other domestic animals. It can be given inregional programs to reduce transmission from dogs or other domesticanimals which serve as a reservoir for the parasite.

4.3.5 Use of DIM-Related Indoles with Antiprotozoal Agents for ProtozoalDiarrheal Disease Due to Crytposporidia or Other Coccidial Protozoa

DIM related indole therapy for Coccidal diarrhea caused typically byCryptosporidium parvum, C. hominis, or Cyclospora cayetanensis is basedon symptoms and identification of the protozoal oocytes in stoolanalysis. In adults, oral DIM-related indoles are typically employed fortreatment using a dose range of 50-200 or 50-400 mg twice daily, aloneor in conjunction with Nitazoxanide 500 mg twice daily in adults.Children use DIM-related indole as a pediatric suspension providing25-150 or 25-300 mg twice daily, alone or in conjunction with asuspension of Nitazoxanide providing 200 mg twice daily. Alternatively,capsules containing a combination of DIM, Artemisinin, and curcumintwice daily can be utilized without Nitazoxanide. These capsules arealso useful in treating Microsporidial infections caused byEnterocylozoon species in immunocompromized patients.

4.3.5.1 Use of DIM-Related Indoles with Antiprotozoal Agents forToxoplamosis

Treatment of toxoplasmic encephalitis consists of administration of oralDIM-related indoles using a dose range of 50-200 or 50-300 mg twicedaily, alone or in conjunction with pyrimethamine and sulfadiazine alongwith folinic acid using established protocols in adults. Children useDIM-related indole as a pediatric suspension providing 25-150 or 25-300mg twice daily, alone or in conjunction with a suspension ofpyrimethamine and sulfadiazine along with folinic acid using establishedpediatric protocols. Alternatively, capsules or pediatric suspensionscontaining a combination of DIM, Artemisinin, curcumin, and piperinetwice daily can be utilized. DIM-related indole at adult doses alone orwith Spiramycin can be used during pregnancy.

4.3.6 Combined Use of DIM-Related Indoles with Anti-Protozoal Agents forTrichomonal Disease.

Trichomonal vulvo-vaginitis in women and urethritis in men is treatedaccording to the present invention by administering a DIM-related indolein addition to standard doses of oral metronidazole (Cudmore S L,Delgaty K L, Hayward-McClelland S F, Petrin D P, Garber G E. Treatmentof infections caused by metronidazole-resistant Trichomonas vaginalis.Clin Microbiol Rev. 2004 October; 17(4):783-93). This can beaccomplished by adding DIM, preferably in an absorption-enhanceddelivery system (U.S. Pat. No. 6,086,915), and administering 100-200 mgorally once or twice daily for 1-2 weeks. Alternatively, DIM isformulated as a vaginal cream alone or in combination with artesunatefor topical administration in infected women. Ideally the vaginal creamis formulated for sustained delivery using site-directed formulationtechnology to produce unit-dose vaginal creams which are retained on themucosa. Preferred embodiments utilize formulation techniques describedin U.S. Pat. Nos. 6,899,890, 6,214,379, 5,730,997, 5,554,380, 5,445,829,5,266,329, and US Patent Applications 20030180366 and 20070154516,20070224226, 20060140990, and 20040062802.

Optionally, the DIM-related indole (50-500 mg) is combined withartemisinin or a derivative such as artesunate, providing 100-300 mg ofartesunate per unit dosage. The combination cream is used every 1-3 daysin conjunction with oral metronidizole or tinidazole at establisheddoses. Alternatively, the DIM-related indole is combined withButoconazole (75-200 mg) or Clindamycin (75-200 mg), with optionaladdition of artemisinin, or artimisinin derivative, to provide atreatment for combined protozoal-bacterial or protozoal-fungalinfections. In a further embodiment, the DIM-related indole (50-500 mg)is combined with extracts incorporating the active ingredient from thefruit pericarp of Sapindus miukorossi for a topical preparation withboth anti-trichomonal and spermicidal activity.

4.3.6.1 Combined Use of DIM-Related Indoles with Anti-Protozoal Agentsand Antibiotics for Mixed Vaginal Infections.

DIM related indoles can be formulated in topical emulsions, some withsustained mucosal retention and release of active agents for use inmixed vaginal infection. These uses include DIM related indoles incombination use with clincamycin for bacterial —yeast infections, oralternatively, combined with both clindamycin and artemisinin derivativefor bacterial-yeast infections. DIM related indoles can be used withartemisinin derivatives for combined trichomonal-yeast infections,combined with butoconazole for combined trichomonal-yeast infections oralternatively, combined with butoconazole and artemisinin derivativesfor combined trichomonal-yeast infections. Preferred embodiments utilizeformulation techniques described in U.S. Pat. Nos. 6,899,890, 6,214,379,5,730,997, 5,554,380, 5,445,829, 5,266,329, and US Patent Applications20030180366 and 20070154516, 20070224226, 20060140990, and 20040062802.Optionally, DIM related indoles can be used in combination with otheruseful antiprotozoal agents including metronidazole, or in combinationwith useful anti-fungal agents, including azole drugs, particularlybutoconazole. In addition, natural products drugs or extracts ofmedicinal plants can be used in combination with DIM related indoles andoptional anti-protozoal, anti-bacterial, or anti-fungal agent. Preferrednatural products for combined use include evodiamine, curcumin, extractsof Sapindus mukorossi, and sodium butyrate. Additionally, DIM relatedindoles, and optional additional anti-protozoal agent, can be combinedwith topically active spermacides for preparations with bothmicrobicidal and contraceptive activities.

4.3.7 Use of DIM-Related Indoles for Intestinal Protozoal Infections inLivestock

In certain embodiments of the invention, formulations include combineduse of DIM-related indoles with antiprotozoal agents to treat intestinalprotozal infections in livestock due to coccidial or neospora protozoalparasites. In particular, for additives to cattle feed to preventcrytosporidal infection associated abortion during breeding, preferredfeed additives utilize, for example, DIM-related indoles, genistein,extracts or derivatives of Artemisia annua, and extracts of Curcumadomestica. Also useful in combination with DIM-related indoles areextracts of Yucca schidigera.

4.3.8 Use of DIM-Related Indoles in Combination with Artemisinin-BasedDrugs.

Typically in human use a twice daily oral dose of 50-250 or 50-600mg/day (1-3 or 1-6 mg/kg/day) of a DIM-related indole in a suitableformulation is taken along with a twice daily oral dose of 25-1000 mg(0.5-10 mg/kg/day) of dihydroartemisinin, artesunate or otherartemisinin-derived drug in a suitable formulation.

In a preferred embodiment, a twice daily oral dose of 50-550 mg/day (1-3mg/kg/day) of DIM-related indole in a suitable formulation is takenalong with a twice daily oral dose of 25-1000 mg (0.5-10 mg/kg/day) ofdihydroartemisinin, artesunate, or other artemisinin-derived drug in asuitable formulation, together with curcumin at a twice daily oral doseof 25-1000 mg (0.5-10 mg/kg/day) and piperine to aid absorption.Typically 20-150 mg of DIM related indole is combined with 25-1500 mg(0.5-10 mg/kg/day) Artemesinin extract and 25-1500 mg (0.5-10 mg/kg/day)of curcumin and 20 mg of piperine 20 mg. Preferably the extract ofpiperine is called Bioperine® (an extract from the fruit of Piper nigrumL (black pepper) or Piper longum L (long pepper) containing 95 percentpiperine; Sabinsa Corporation, Piscataway, N.J.). The DIM-relatedindole, artemisinin-related drug, curcumin and piperine are preferablycontained in the same capsule or tablet to facilitate combined use.

In a second preferred embodiment, the DIM-related indole and artemisininderivative (preferably artesunate) is formulated using site-directedformulation technology to produce unit-dose vaginal or rectalsuppositories/creams which are retained on the mucosa for sustaineddelivery. Related, optional embodiments utilizes formulation techniquesdescribed in U.S. Pat. Nos. 6,899,890, 6,214,379, 5,730,997, 5,554,380,5,445,829, 5,266,329, and US Patent Applications 20030180366 and20070154516, 20070224226, 20060140990, 20040062802, and 20020044961.

For severe protozoal disease as seen with cerebral malaria, Africansleeping sickness, acute Chaga's disease, and visceral Leismaniasis, theDIM-related indole is preferably give intravenously. DIM is given in asuitable intravenous suspension or emulsion to deliver 2-15 mg/kg perdose every 8 to 12 hours. DIM may be administered with quinine inmalaria, with eflornithine, pentamidine, melarsoprol, nifurtimox, orbenznidazole, for tryanosomiasis, and with pentavalent antimony,amphotericin B, or miltefosine in Leishmaniasis.

For life threatening conditions, intravenous DIM-related indoles wouldbe administered with or without DIM suppositories and/or additionalaerosolized DIM. Oxygen therapy is added to potentiate parasiteclearance using DIM-related indoles.

4.4 Administration and Dosage

In certain embodiments, certain combinations of DIM-related indoles,e.g., DIM, and one or more known antiprotozal agents in parenteraldelivery systems, oral delivery systems, rectal suppositories, vaginalcreams, or by simultaneous delivery by multiple routes providestherapeutic efficacy are believed to provide more than the additiveefficacy of each agent used alone at maximal dose. Therefore, methodsinvolving combined use of a DIM-related indole and a known antiprotozalagent at less than their maximal doses increase both the safety andefficacy of DIM-related indoles and antiprotozal agents in selectedprotozoal infections.

Improved efficacy results in a shorter duration of required therapy thanwith individual agents used alone. Combined use can allow a reduction indose or shortening of the period of high dose treatment. Combined usecan improve the long term therapeutic result with a lower rate ofrecrudescence with renewed appearance and growth of surviving parasites.Combined use with lowered dose and duration of use can minimizetoxicity.

In methods involving the oral use of one or more DIM-related indoles,e.g., DIM, and one or more known antiprotozal agents, the oral deliveryof indole is facilitated and accomplished according to formulations andmethods described in U.S. Pat. No. 6,086,915, incorporated by referenceherein in its entirety. In one embodiment, DIM, or a DIM-related indole,is processed with phosphatidyl choline. Alternatively, oral and rectalbioavailability of DIM-related indoles are improved using other meansincluding particle size reduction, complexation withphosphatidylcholine, and formation into rapidly dissolving particles andnanoparticles.

The treatment of protozoal disease with an oral DIM-related indole,e.g., DIM, is facilitated by oral, sublingual, intravenous, rectal,vaginal, transdermal, and intra-lesional application of DIM-relatedindoles in specific relative doses with simultaneous administration of aknown antiprotazoal agent. These therapies include production oftinctures, liposomes, creams, or rectal/vaginal suppositories, emulsionsfor intravenous use, and injectable suspensions to deliver synergisticamounts of these agents. Injectable formulations include cyclodextrincomplexed DIM-related indoles and liposome encapsulated DIM-relatedindoles.

For oral use, DIM is used, preferably formulated for enhanced absorptionin a daily dose of 0.5-12 mg/kg per day. Oral DIM is optionally combinedwith other oral agents for malaria, trypanosomiasis, leishmaniasis,crytosporidiosis, and toxoplasmosis using standard doses of theadditional agents.

For intravenous use, DIM is used, preferably formulated as anintravenous suspension or emulsion, in a daily dose of 0.5-15 mg/kg perday. Intravenous DIM is optionally combined with other intravenous andoral agents for malaria, trypanosomiasis, leishmaniasis,crytosporidiosis, and toxoplasmosis using standard doses of theadditional agents.

For rectal use, DIM is preferably formulated for enhanced rectalabsorption in suppositories in a daily dose of 0.5-12 mg/kg per day. DIMis optionally combined with other rectal agents for malaria (artesunate,artemether), trypanosomiasis (eflornithine, nifurtimox), leishmaniasis(miltefosine), crytosporidiosis (artesunate, artemether), andtoxoplasmosis (pyrimethamine and sulfadiazine) using standard doses ofthe additional agents.

For vaginal or rectal use the DIM-related indole is formulated usingsite-directed formulation technology to produce unit-dose vaginal orrectal suppositories/creams which are retained on the mucosa forsustained delivery. Preferred embodiments utilize formulation techniquesdescribed in U.S. Pat. Nos. 6,899,890, 6,214,379, 5,730,997, 5,554,380,5,445,829, 5,266,329, and US Patent Applications 20030180366 and20070154516, 20070224226, 20060140990, 20040062802, and 20020044961.Optionally, the DIM-related indole is combined with artemisininderivative (e.g., artesunate), butoconazole, tinidazole, niridazole,nitazoxanide, or miltefosine,

4.5 Pharmaceutical/Nutraceutical Compositions

Pharmaceutical/Nutraceutical Dosage Forms for DIM-related indoles:Multi-application DIM-related indole containing particles aremanufactured by various techniques including spray drying, spraycooling, selective precipitation, crystallization and other particleforming methods. The resulting particles are used in the manufacture ofthe following dosage forms, some of which are described in U.S. Pat. No.6,086,915, incorporated by reference herein in its entirety.

I. Spray Dried Microencapsulated Solid Dispersions

-   -   1. TPGS/phosphospholipid spray-dried particles. Production of        absorption-enhanced DIM-related indole particle formation is        provided in U.S. Pat. No. 6,086,915.    -   2. Liquid emulsions using TPGS/phosphospholipid spray-dried        particles. Production of emulsions for oral use utilizes        absorption-enhanced DIM-related indole particle formation is        provided in the U.S. Pat. No. 6,086,915.    -   3. Flavored DIM granules for oral use (Chocolate, Orange        “sprinkles”). Production of flavored granules for oral use        utilizes absorption-enhanced DIM-related indole particles        (DIM/TPGS) as provided in U.S. Pat. No. 6,086,915. Production        steps include dry mixing DIM/TPGS particles with maltodextrin        granules, addition of flavoring particles and granulation using        a standard fluid bed granulator.

II. Spray Dried Polymer Based Solid Dispersions

Production techniques for DIM-related indoles may utilize thosedescribed in U.S. Patent Application No. 20030072801, entitled“Pharmaceutical compositions comprising drug and concentration-enhancingpolymers,” herein incorporated by reference in its entirety. Inparticular production involves the following dissolution enhancingpolymers, used with and without lipid stabilizers:

-   -   1. Polymer included: Hydroxy Propyl Methylcellulose    -   2. Polymer: Hydroxy Propyl Cellulose

III. Cyclodextrin Based Formulations

Examples of manufacturing techniques are described in U.S. Pat. No.4,877,778 and U.S. Patent Applications Nos. 20040053888; 20030073665;and 20020068720, each of which is herein incorporated by reference inits entirety. Using cyclodextrin loading production techniques toincorporate DIM-related indoles the following final formulations areproduced:

-   -   1. Dry particle complex for oral use    -   2. Intravenous emulsion    -   3. Parenteral emulsion

IV. Nanoparticle-Based Dispersions

Examples of manufacturing techniques are described in U.S. Pat. Nos.6,288,040; 6,165,988; 6,117,454; and U.S. Patent Application PublicationNo. 20030032601; each of which is incorporated by reference in itsentirety. Using nanoparticle production techniques to incorporateDIM-related indoles the following final formulations are produced:

-   -   1. Dry particle complex for oral use.    -   2. Intravenous emulsion    -   3. Parenteral emulsion

V. Liposome Based Formulations

Examples of manufacturing techniques are described in U.S. Pat. Nos.4,906,476; 5,006,343; and U.S. Patent Application Publication No.20030108597. Using liposome production techniques to incorporateDIM-related indoles the following final formulations are produced:

-   -   1. Dry particle complex for oral use    -   2. Intravenous emulsion    -   3. Parenteral emulsion

4.5.1 Oral Combined Products

Combined formulations for oral use include DIM-related indole,optionally formulated for enhanced absorption, combined with one or moreadditional anti-protozoal compounds and optionally include additionalabsorption enhancers. Examples of preferred antiprotozoal compoundsinclude artemisinin; dihydroartemisinin; artemethe; artesunate;atovaquone; diaminopyrimidines, especially amodiaquine, amphotericin,clindamycin, eflornithine, fumagillin; the 8-hydroxyquinolines,chloroquine, mefloquine, halofantrine, lumefantrine, geldanamycin,iodoquinol (diiodohydroxyquin) and clioquinol (iodochlorhydroxyquin);the 2-nitroimidazoles including Etanidazole and Benznidazole;doxycycline; melarsoprol; metronidazole; miltefosine; nifurtimox;nitazoxanide; paromomycin; pentamindine; sodium stibogluconate andrelated antimonials; suramin; pyrimethamine; proguanil (chloroguanide);spiramycin; sulfadoxine; sulfonamides including trimetoprim; sulfones;and tetracyclines; quinine derived from the bark of the South Americanchinchona tree; 6-gingerol and/or 6-paradol (Surh et al., 1999, JEnviron Pathol Toxicol Oncol. 18(2): 131-9); and selected flavonoids andderivatives (Tasdemir et al., 2006, Antimicrob Agents Chemother.50(4):1352-64). Other anti-protozoal compounds include, but are notlimited to, curcumin, an extract of Curcuma domestica, leutiolin,selenium compounds, especially methylselenic acid, resveratrol,including an extract of Polygonium cuspidatum, silibinin, an extract ofSilybum marianum, apigenin, deguelin, extracted from various plantsources including Munduelea sericea, Evodiamine, ursolic acid,Andrographolide, Dehydro-Andrographolide, Deoxy-Andrographolide,Brassinin, Caffeic acid, Capsanthin, Capsaincin, Chelerythrine Chloride,Cromolyn sodium, Allyl Disulfide, Diallyl disulfide, Diallyl sulfide,Diallyl trisulfide, Dibenzoylmethane, Ebulin 1, Ellagic acid, Ferulicacid, 18β-Glycyrrhetinic Acid, Glycyrrhizic acid ammonium salttrihydrate, Honokiol, 5-Hydroxy-L-tryptophan, Hypericin, Hypocrellin A,Idebenone, luteolin, D-Limonene, Limonin, Limonin Glucoside, DL-α-Lipoicacid, Melatonin, Perillyl Alcohol, Phenylbutyrate, Phenylethyl3-methylcaffeate, Phenylethy 14-methylcaffeate, Phenyl isothiocyanate,Phytic Acid, Rosmarinic acid, Rutaecarpine, sulforaphane, L-Threonine,Trichostatin A, aspirin, salycylamide. Absorption-enhancing agents canbe additionally added including, but not limited to, Vitamin-Epolyethyleneglycol succinate (TPGS), piperine, limonine, D-Limonene,and/or polyethyleneglycol. In addition, orally active immunepotentiating agents can be utilized in addition to anti-protozoal agentsincluding Vitamin K3, N-Acetyl-L-Cysteine, Zinc citrate, or Zincgluconate.

The DIM, or a DIM-related indole, together with one or moreantiprotozoal compounds and optionally, an antiprotozoal plant extractcan also be added to selected foods as fortified, “functional” foods.Fortified foods include “medicinal foods” which require use under adoctor's care and “functional foods” available to consumers asunregulated specialized foods. Such uses in fortified or “functional”foods typically apply to Food Bars, Drink Mixes, Vegetable Juices, PastaMixes, Dry Cereal, Meal Replacement Powders, Tea mixes, and Baked Goods.Such uses require specialized production with the dose of DIM inaccordance with principles of Generally Regarded As Safe (GRAS) foodingredients. These typically include drink mixes, meal replacementpowders, food bars, and candies. Formulations include DIM, or aDIM-related indole, and one or more of the following antiprotozoalcompounds: artemisinin, genistein, curcumin, and resveratrol. Thecompositions of DIM, and DIM-related indoles, of the present inventionare also utilized as added ingredients to fortified foods to facilitateconvenient and regular consumption to prevent or control chronicprotozoal parasite infection. Antiprotozoal fortified foods with DIM areused in periodic “cleansing” programs, where special diets andintermittent fasting is use to reduce intestinal parasite infection. Insuch applications DIM is added to food products or mixes alone or incombination with selected antiprotozoal compounds, particularlyartemisinin, curcumin, and isoflavones like genistein.

Food Bar Products are produced according to the present inventionaccording to known manufacturing and baking practices. Detailed of foodbar composition and manufacturing techniques useful with DIM,DIM-Related Indoles, and DIM combined with selected antiprotozoalcompounds are specified in U.S. Patent Application Publication Nos.20030068419 entitled “Food bar compositions” and 20020168448 entitled“Nutritional food bar for sustained energy”.

Drink Mix Products are produced according to the present inventionaccording to known manufacturing practices. Detailed drink mixcomposition and manufacturing techniques useful with DIM, DIM-RelatedIndoles, and DIM combined with selected antiprotozoal compounds arespecified in U.S. Pat. No. 6,599,553 by Kealey et al., entitled “Drydrink mix and chocolate flavored drink made therefrom”.

In preferred embodiments, DIM is incorporated in fortified foods, suchas drink mixes and food bars, during food production using a particulateform of DIM that is formulated for enhanced absorption (BioResponse-DIM[BioResponse, LLC, Boulder, Colo.]). Artemisin is added as a powderedextract of Artemisia annua (Artemisin [Allergy Research Group, Alameda,Calif.]). Genistein is added as a powdered formulation of pureisoflavone (Bonistein [DSM Nutritional Products]). Typically, the DIM isprovided in a dose of 10-75 mg/serving (40-300 mg/serving ofBioResponse-DIM). Artemisinin is provided in a dose of 50-300mg/serving. Genistein is provided in a dose of 25-100 mg/serving asBonistein.

4.5.2 Mucosally Targeted Formulations

Mucosally targeted formulations includes specialized emulsions ofDIM-related indoles, optionally combined with known anti-protozoalagents which are retained on and penetrate the targeted mucosa for longperiods of time. This includes specialized formulations for oral,esphogeal, rectal, vaginal, and vulvar mucosa. This formulationtechnology provides a preferred way of treating Trichomonal infection,Candidal infection, and Crytosporidial infection. For vaginal or rectaluse the DIM-related indole is formulated using site-directed formulationtechnology to produce unit-dose vaginal or rectal suppositories/creamswhich are retained on the mucosa for sustained delivery. One relatedembodiment utilizes formulation techniques described in U.S. Pat. Nos.6,899,890, 5,554,380, 5,266,329, and US Patent Applications 20030180366and 20070154516. Optionally, the DIM-related indole is combined withartemisinin derivative (eg, artesunate), butoconazole, clindamycin,tinidazole, niridazole, nitazoxanide, or miltefosine,

4.5.3 Hepatic Targeted Formulations

Hepatic targeting includes intravenous emulsions which are concentratedin liver tissue with or without lipids and, optionally includinganti-protozoa-specific antibodies. Concentration in hepatic tissueprovides an advantage in treating the hepatic stage of malaria.

-   -   1. Phospholipid complexed intravenous emulsions    -   2. Cyclodextrin-based intravenous emulsions    -   3. Intravenous suspension complexed with Anti-protozoal        monoclonal antibodies

4.5.4 Leukocyte Targeted Formulations

Leukocyte targeting includes intravenous emulsions which areconcentrated in leukocytes with or without lipids andAnti-protozoa-specific antibodies. Concentration in leukocytes providesan advantage in treating the leukocyte stage of trypanosomal andleishmanial infections.

-   -   1. Phospholipid complexed intravenous emulsions    -   2. Cyclodextrin-based intravenous emulsions    -   3. Intravenous suspension complexed with Anti-Protozoal        monoclonal antibodies

4.5.5 DIM Tinctures and Suspensions

Tincture preparation allows a simple absorbable liquid formulation ofDIM-related indole to be prepared for use in a rural setting. A mixtureof DIM-related indole is manufactured with optional, additionalanti-protozoal compound using ethanol as a solvent to provide varioustincture formulations. Methods for making tintures appropriate foranti-protozoal compounds are described by Sweet in U.S. Pat. No.7,033,619, entitled “Method for making herbal extracts usingpercolation” and methods specific for Atemesinin tinctures described inU.S. Pat. No. 4,952,603.

-   -   1. Oral tinctures prepared with ethanol or hexanol    -   2. Parenteral tincture prepared with ethanol or hexanol    -   3. Microcrystalline suspension for oral use    -   4. Parenteral microcrystalline suspension    -   5. Sustained release parenteral suspension

Pharmaceutical compositions according to the present inventionpreferably comprise one or more pharmaceutically acceptable carriers andthe active constituents, e.g., a DIM-related indole alone, or aDIM-related indole and one or more known antiprotozal agents. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe other ingredients of the composition and not deleterious to therecipient thereof.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; a glidant, such as colloidal silicon dioxide; asweetening agent, such as sucrose or saccharin; and/or a flavoringagent, such as peppermint, methyl salicylate, or orange flavoring.

It will be appreciated that the amounts of DIM or other DIM-relatedindole and/or one or more known antiprotozal agents, required for thetreatments disclosed herein will vary according to the route ofadministration, the severity of the protozoal disease, age, and medicalhistory of the subject, the galenic formulation of the pharmaceuticalcomposition, etc.

Preferably, the DIM used in the invention has been processed to enhancebioavailability, as is described in U.S. Pat. No. 6,086,915,incorporated herein by reference in its entirety; however any suitablepreparation of pure diindolylmethane can be used in the methods andcompositions of the invention. Optionally, additional absorptionenhancing agents active with DIM-related indole such as grapefruitextracts or extract of black pepper providing extracts of piperine canbe included.

In general, a suitable (therapeutically effective) amount ofDiindolylmethane is preferably administered in an absorption enhancingformulation, as described in U.S. Pat. No. 6,086,915, at 25-750 mg perday as a suspension of microparticles in a starch carrier matrix.Structurally-related, synthetically-derived, substituteddiindolylmethane's, as described by Jong (U.S. Patent ApplicationPublication No. 2004/0043965) are administered according to the presentinvention in an acceptable formulation for oral administration in a doseof 10-400 mg/day. Preferably, these substituted diindolylmethanes areadministered in an absorption-enhanced formulation at a dose of 50 to250 mg/day. The actually administered amounts of DIM or a substituteddiindolylmethane may be decided by a supervising physician. TheDIM-related indole of the invention is preferably administered incombination with one or more known antiprotozal agents administered byeither oral, rectal, or parenteral routes.

Therapeutic formulations include those suitable for parenteral(including intramuscular and intravenous), topical, oral, vaginal,rectal or intradermal administration. Thus, the pharmaceuticalcomposition may be formulated as tablets, pills, syrups, capsules,suppositories, ophthalmic suspension, formulations for transdermalapplication, powders, especially lyophilized powders for reconstitutionwith a carrier for intravenous administration, etc. Oral administrationfor DIM is the most preferred route.

Therapeutic formulations suitable for oral administration, e.g., tabletsand pills, may be obtained by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be prepared bymixing phytochemicals, and compressing this mixture in a suitableapparatus into tablets having a suitable size. Prior to the mixing, theDIM-related indole or one or more antiprotozoal agents may be mixed witha binder, a lubricant, absorption enhancer, an inert diluent and/or adisintegrating agent.

In a preferred embodiment, the DIM-related indole is mixed with abinder, such as microcrystalline cellulose, and a surfactant, such assodium lauryl sulphate until a homogeneous mixture is obtained.Subsequently, another binder, such as polyvinylpyrrolidone (polyvidone),is transferred to the mixture under stirring with a small amount ofadded water. This mixture is passed through granulating sieves and driedby desiccation before compression into tablets in a standard tabletingapparatus.

A tablet may be coated or uncoated. An uncoated tablet may be scored. Acoated tablet may be coated with sugar, shellac, film or other entericcoating agents.

Therapeutic formulations suitable for parenteral administration includesterile solutions or suspensions of the active constituents. An aqueousor oily carrier may be used. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Formulations for parenteral administrationalso include a lyophilized powder comprising phytochemical that is to bereconstituted by dissolving in a pharmaceutically acceptable carrierthat dissolves said phytochemical. Parenteral administration alsoincludes a stable emulsion of DIM designed for intravenous use. Ideally,the emulsion prevents the early removal of DIM from the circulation dueto early uptake by the reticulo-endothelial system allowing maximalcellular concentration of DIM in parasite-infected cells or tissue.

When the pharmaceutical composition is a capsule, it may contain aliquid carrier, such as a fatty oil, e.g., cacao butter.

Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. These compositions cantake the form of solutions, suspensions, emulsion, tablets, pills,capsules, powders, sustained-release formulations and the like. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides or nerolidol, a sesquiterpene.

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 1987, 14:201;Buchwald et al., Surgery 1980, 88:507; Saudek et al., N. Engl. J. Med.1989, 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.1983, 23:61; see also Levy et al., Science 1985, 228:190; During et al.,Ann. Neurol. 1989, 25:351; Howard et al., J. Neurosurg. 1989, 71:105).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In one embodiment of the pharmaceutical composition according to theinvention, the DIM-related indole and one or more known antiprotozalagents are comprised as separate entities. The entities may beadministered simultaneously or sequentially.

The invention also provides a pack or kit comprising one or morecontainers filled with one or more of the ingredients of thecompositions of the invention. This includes the combination of capsulesfor oral use and rectal suppositories. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

A number of references have been cited, the entire disclosures of whichare incorporated herein by reference.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

5. EXAMPLES 5.1 Example 1 Manufacture of Processed DIM for Enhanced OralBioavailability

Preparation of Processed Diindolylmethane is Accomplished According tothe steps outlined in U.S. Pat. No. 6,086,915, herein incorporated byreference in its entirety. Briefly, this included mixture of about10-40% by final weight of Diindolylmethane with about 10-40% by finalweight of vitamin E polyethylene glycol 1000 succinate (Vitamin-E-TPGS,Eastman Chemical), 2-20% by final weight, phosphatidyl choline(Phospholipon 50G, Rhone Poulenc) and 15-30% by final weight hexanol.This mixture is made homogeneous by mixing. The homogeneous mixture ofindoles and other oil soluble substituents listed above is added to asolution of modified starch in water (Capsul Starch from NationalStarch, Inc.). The starch component forms from 30-70% of the final dryweight of the product. The well dispersed final combined mixture is thensubjected to spray drying. The resultant product is a fine powdercontaining Diindolylmethane contained within the starch particles.

5.2 Example 2 Manufacture of Capsules Containing Diindolylmethane andOptional Antiprotozoal Agent(s)

Capsules containing 150-300 mg of processed Diindolylmethane, asproduced according to the steps described in example 6.1, are made bymixing the processed Diindolylmethane with microcrystalline celluloseand placing the mixed powder into opaque gelatin capsules.

Capsules containing the combination of about 150 mg of processed DIM(providing 50 mg of DIM) and 200-500 mg of Artemisinin, and/or otherArtemisinin derivatives are made by mixing the processed DIM, andArtemisinin with microcrystalline cellulose or rice flour excipient andplacing the mixed powder into opaque gelatin capsules. Alternatively,capsules or tablets with about 150 mg of processed DIM providing 50 mgof DIM and 200-500 mg of Artemisinin, or other Artemisinin derivatives,Curcumin 100-200 mg, and piperine 20 mg are made with microcrystallinecellulose or rice flour excipient and by placing the mixed powder intoopaque gelatin capsules.

5.3 Example 3 Manufacture of Flavored, Pediatric Suspensions ofDIM-Related Indoles

DIM-related indoles are incorporated into pediatric suspensionsmanufacture as powdered mixtures to be re-constituted with water priorto use in patients. The suspension products have the advantages of longshelf life, stability and flavoring for taste masking to improvepalatability.

A DIM-related indole containing suspension is made using establishedmanufacturing techniques as described in U.S. Pat. No. 6,586,012 by Yuet al. issued Jul. 1, 2003, and titled, “Taste masked pharmaceuticalliquid formulations”. When reconstituted, the pediatric suspension has aDIM related indole concentration of 15-30 mg/ml of suspension.Typically, 3-10 mg/kg/dose of DIM related indole is administered twicedaily. Alternatively, DIM-related indole pediatric suspensions aremanufactured using techniques described by Kulkarni et al. in U.S.Patent application No. 20050136114, published June 23, 200, and titled,“Taste masked pharmaceutical compositions comprising bitter drug and pHsensitive polymer”.

In a preferred embodiment the DIM-related indole is formulated into apediatric suspension in combination with an artemisinin derivative, suchas artesunate, curcumin and piperine utilizing taste masking.

5.4 Example 4 Manufacture of DIM with Artemisinin Derivative in aSuppository for Vaginal or Rectal Administration

In a heated vessel, 90 grams cetostearyl alcohol (Alfol 16/18, Vista)mixed with 10 cc Grapefruit Oil (Aldrich Chemical) was heated to 100° C.to which 20 gms of microcrystalline DIM, 5 or 10 gms of Artesunate (LKTLabs, St. Paul, Minn.), were added with constant mixing to form a hotslurry. Alternatively, 90 grams cetostearyl alcohol (Alfol 16/18, Vista)is heated to 100° C. to which 10 gms of microcrystalline DIM is mixedand to which is added in a second vessel 400 gms of IV Novata(Semi-synthetic Glyceride Suppository Base, Ashland Chemicals) waswarmed to 40° C. with constant mixing. The well mixed slurry from thefirst vessel was added with continued mixing to the second vessel. Thehomogenized molted suppository material was formed into suppositories of2 g each and cooled. Glyceryl monsterate 10-50 g was added to the moltenmixture as needed to increase the firmness of the final suppositories.Optionally, 50-100 mg of artesunate or arthemether is added persuppository for children, and 200-300 mg of artesunate or artemether isadded per suppository for adults.

Other methods of producing suppositories are well know in the art asdescribed in U.S. Pat. No. 4,164,573 by Galinsky et al., issued Aug. 14,1979 and titled, “Composition and method for making a suppository forintroducing a hypoglycemic agent into a mammal”. Use of this methodprovides a technique to manufacture a mixture of DIM andArtemisinin-related compound which is then evaporated into a semisolidmass and then shaped into suppository. In a preferred product format thesuppository would contain 50-100 mg of DIM and 50-100 mg of artesunatefor children, and 100-300 mg of DIM and 200-300 mg of artesunate foradults. Optionally, Vitamin E TPGS (Eastman Chemical) or nerolidol, asesquiterpene, are added to suppository formulations as drug penetrationenhancers.

5.5 Example 5 Manufacture of Sustained-Release Combination Formulas forVaginal or Rectal Administration

Mucosally targeted formulations includes specialized emulsions ofDIM-related indoles, optionally combined with known anti-protozoalagents which are retained on and penetrate the targeted mucosa for longperiods of time. This includes specialized formulations for oral,esphogeal, rectal, vaginal, and vulvar mucosa. This formulationtechnology provides a preferred way of treating Trichomonal infection,Candidal infection, and Crytosporidial infection. For vaginal or rectaluse the DIM-related indole is formulated using site-directed formulationtechnology to produce unit-dose vaginal or rectal suppositories/creamswhich are retained on the mucosa for sustained delivery. One relatedembodiment utilizes formulation techniques described in U.S. Pat. Nos.6,899,890, 5,554,380, 5,266,329, and US Patent Applications 20030180366and 20070154516. Optionally, the DIM-related indole is combined withartemisinin derivative (eg, artesunate), butoconazole, clindamycin,tinidazole, niridazole, nitazoxanide, or miltefosine,

5.6 Example 6 Manufacture of Cyclodextrin Complex Formulations withDIM-Related Indoles for Improved Bio-Delivery

Introduction: As poorly soluble drug agents, DIM-related indoles, ingeneral, require solubility enhancing formulation steps which arebio-compatible for parenteral and improved oral drug delivery.Parenteral formulations for intramuscular, intravenous, and pulmonaryaerosol delivery benefit from complexation with various cyclodextrins(alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin,and sulfobutylether-beta-cyclodextrin).

Methods: Specific formulations and formulation steps are developedutilizing cyclodextrins. Formulations are based on methods andobservations that molecules containing indole rings successfully complexwith cyclodextrins providing a subsequent solubility advantatage overthe indole alone (Cao et al., 2000, Chemosphere 40:1411-6). Therefore,prototype formulation utilizing microcrystalline DIM, compatible solventsystems, with and without lipid stabilizers are manufactured using spraydrying technology. Dry particle products are appropriate for suspensionin aqueous vehicles for intramuscular or intravenous drug delivery.

Preferred cyclodextrins utilized are:

-   -   1. β-cyclodextrin, which is generally more applicable for the        complexation of hydrophobic molecules. It is anticipated that 2        molecules of β-cyclodextrin will be needed per molecule of DIM.    -   2. Hydroxypropyl β-cyclodextrin, which is known to be very        soluble, on the order of 30% and more.    -   3. Sulfobutyl β-cyclodextrin (trade-name Captisol®). Captisol        has a molecular weight of ˜2200 mg/mmol so at 2:1 cyclodextrin        to DIM, the amount of DIM that can be put into solution will be        considerably higher than the known solubility of DIM.

Sample preparations to be undertaken:

Pharmaceutically acceptable solvents will be utilized to form solutionsfor spray drying with Hydroxypropyl β-cyclodextrin and Sulfobutylβ-cyclodextrin. Complexes of each of the β-cyclodextrins with DIM willbe prepared with a slight excess of the cyclodextrin and spray dried toproduce approximately 10 to 20 grams of each formulation. Furtherformulation suitable for intravenous, intramuscular and pulmonaryaerosol use will utilize published manufacturing techniques (Steckel etal., 2004, Int J. Pharm. 278:187-95).

Testing of prepared DIM-cyclodextrin formulations:

Prepared samples will be analyzed as to amorphous crystal structure andstability using standard techniques (Rodriguez-Spong et al., 2004, AdvDrug Deliv Rev. 56:241-74). Testing of prepared formulations willinclude release testing of DIM in simulated gastric acid. In vivorelease and bioavailability testing in animal and human models willutilize plasma DIM assays as described in U.S. Patent ApplicationPublication No. 20030096855.

Conclusions: Cyclodextrin DIM Spray-dried formulations are expected todemonstrate shelf stability, form stable suspensions in 5% dextrosesolutions for intravenous administration, and stable suspensions in 0.9%NaCl for intramuscular, parenteral administration.

5.7 Example 7 Sterile Intravenous Microemulsions of DIM for Use inConjunction with Protozoal Parasite Treatment

Stable microemulsions of DIM, designed for intravenous use, aredeveloped to provide a convenient means of administering DIM to achievehigh tissue concentrations of DIM quickly and at a predictable time.This use facilitates the use of DIM in anti-protozoal therapy. Inaddition, microemulsions of DIM can be used in conjunction with otheranti-protozoal agents. In alternative embodiments, DIM analoguesincluding imidazolelyl-3,3′-diindolylmethane, including nitrosubstituted imidazolelyl-3,3′-diindolylmethanes and DIM derivativeSR13668 (Stanford Research Institute) can be used in protozoal parasiteinfections.

The low solubility of DIM in both water and lipid requires developmentof a specialized micro-emulsion that utilizes phospholipids to optimizethe solubility of DIM and improve the stability of the microemulsion. Toprepare the micro-emulsion Ethyl oleate (EO), Phosphatidyl Choline (PC)(from egg yolk), and calcein, are purchased from Sigma-Aldrich, Inc (St.Louis, Mo.). Distearoyl-phosphatidylethanolamine-N-poly(ethyleneglycol)2000 (DSPE-PEG) is purchased from Avanti Polar Lipids (Alabaster, Ala.).

Using a modification of the method of Yu et al. (Yu et al., 1993, Int.J. Pharm. 89:139-146), the microemulsion is manufactured as follows: 160grams of EO and 60 grams of PC are dissolved in 1 liter pure ethanol. 24grams of microcrystalline DIM (mean particle size 0.25 micron) is addedand dissolved in this “oily phase”. 20 grams of DSPEG-PEG is thendissolved in 500 cc of USP water (Aqueous phase). The oily ethanolicsolution (oily phase) with the dissolved DIM is then slowly added intothe DSPE-PEG solution (aqueous phase) under moderate magnetic stirring.The aqueous phase immediately turns milky with opalescence as the resultof the microemulsion produced. The microemulsion is then subjected tolow pressure at 360 mm Hg and maintained at 50° C. The low pressure isused to concentrate the emulsion through removal of the ethanol and aportion of the water. Using an infrared absorption assay to determinethe DIM content of the microemulsion, a final concentration of DIM of7.5 mg/ml is established. Sodium hydroxide is added to increase the pHto the 5.0-7.5 range.

Using this manufacturing technique emulsions of DIM are prepared andsubjected to stability testing to demonstrate that the particle sizewithin the emulsion remained between 150 and 200 nm. The productiontechnique results in a micro-emulsion with % weight ranges of thecomponents in the following preferred ranges:

Component Approx % Weight DIM 0.05-0.1  Lipids (EO:PC:DSPE-PEG; 8:3:1)45-28 Water 50-70 Ethanol 1-2

Alternatively, an ethanol-free production method can be utilized toproduce a stable micro-emulsion of DIM or DIM derivatives and analogues,using Lipofundin MCT B (Braun Melsungen AG, Melsungen, Germany), apreformed basic emulsion, and high pressure homogenization ofmicrocrystalline DIM. This method utilizes jet-milled DIM, with particlesize reduced to 0.1 micron average diameter (performed by MicronTechnologies, Inc., Exton, Pa.). Using this technique 700 mg of 0.1micron diameter DIM crystals are homogenized in 100 cc Lipofundin usingequipment and methods as described (Akkar et al., 2003, Eur J PharmBiopharm. 55:305-12). This results in a stable lipid-basedmicro-emulsion with particle size less than 200 nm and a DIM content of7 mg/cc of the emulsion.

5.8 Example 8 Sterile Liposome-Encapsulated DIM for Oral, Rectal, and/orIntravenous for Use in Conjunction with Antiprotozoal Therapy

Liposomes are microscopic vesicles composed of a phospholipid bilayerthat encapsulate active agents for specialized delivery to specifictissues. In certain embodiments, liposome encapsulated DIM formulationsare developed to provide increased concentration of DIM in hepatic andpulmonary tissue in anti-protozoal therapy. Manufacturing techniques forDIM Liposomes are developed based on the published liposomemanufacturing techniques as described in U.S. Pat. Nos. 4,906,476;5,006,343; and U.S. Patent Application Publication No. 20030108597, eachof which is incorporated by reference herein in its entirety. Thepreferred techniques for producing DIM liposomes are those that resultin liposomes which accumulate in lung and respiratory epithelial tissue.

Liposomes are formulated utilizing N-(carbonyl-methoxy-polyethyleneglycol 2000)-1,2 disteaoyl-sn-glycero-3-phosphoethanolamine sodium salt(MPGEG-DSPE) (2-4 mg/ml); fully hydrogenated soy phosphatidylcholine(HSPC) (2-11 mg/ml); and cholesterol (1-4 mg/ml). Each 30 ml vialproduced contains 30-60 mg of DIM-related indole at a concentration of1-2 mg/ml.

DIM Liposomes can be utilized in hospitalized cases of protozoal diseaseevery 8 to 12 hours. DIM Liposomes are preferably administeredintravenously or per rectum if intravenous access is not possible.

5.9 Example 9 Apoptosis Promoting Activity of DIM and AntiprotazoalCompounds in a Cell Culture Models of Malaria, Trypanosomiasis,Leishmaniasis, Cryptospridiosis, Trichomonaiasis and Blastocystosis

Introduction:

Cell culture techniques relevant to human protozoal infections includemethods developed to test antiprotozoal agents in models of the bloodstage of malaria, models of macrophage infection with amastigotes ofLeishmania, models of macrophage infection with Trypanosomes, models ofenterocyte infection with Cryptosporidium, models of infection withTrichomonas, and models of enterocyte infection with BlastocystisHominis. Preferred methods allow culture conditions and testing for thecell death response induced by the anti-protazoal agent(s) attributableto apoptosis. Testing of a DIM-related indole alone to establish theminimum effective concentration in culture media and the concentrationfrom reduction of infected cell number by 50% (EC₅₀) is conducted. Thecombination of one or more antiprotozoal agents in combination with aDIM-related indole is used to establish additive or synergistic activityaccording to established methods (Nduat, 2006, Acta Trop. 97(3):357-63).

The relevant in vitro culture and endpoint assay methods for use withDIM-related indoles alone and in combination are listed in the followingchart.

Parasite Culture Methods Assay Methods (Ref) Plasmodium Species P.falciparum in human RBC's Giemsa-staining RBC's (1, 2) TrypanosomeSpecies T. Cruzi or T. brucie in media Alamar Blue Florometric dye (3,4) Leishmania Species L. donovani in media Alamar Blue Florometric dye(4) Cryptosporidium Species H69 Human Bile Duct Cells DAPI staining forapoptosis (5) Blastocystis Hominis Culture of B. Hominis in media. Phasecontrast microscopy and flow cytometry (6) Trichomonas vaginalis Cultureof T. Vaginalis in media Aerobic and anerobic culture with invertedphase contrast microscopy (7) References for In Vitro Methods: (1).Kumar R, Musiyenko A, Barik S. The heat shock protein 90 of Plasmodiumfalciparum and antimalarial activity of its inhibitor, geldanamycin.Malar J. 2003 Sep. 15; 2: 30. (2). Trager W, Jensen J B. Human malariaparasites in continuous culture. 1976. J Parasitol. 2005 June; 91(3):484-6. (3). X. Verma N K, Dey C S. Possible mechanism ofmiltefosine-mediated death of Leishmania donovani. Antimicrob AgentsChemother. 2004 August; 48(8): 3010-5 (4). Tasdemir D, Kaiser M, Brun R,Yardley V, Schmidt T J, Tosun F, Ruedi P. Antitrypanosomal andantileishmanial activities of flavonoids and their analogues: in vitro,in vivo, structure-activity relationship, and quantitativestructure-activity relationship studies. Antimicrob Agents Chemother.2006 April; 50(4): 1352-64. (5). Chen X M, Gores G J, Paya C V, LaRussoN F. Cryptosporidium parvum induces apoptosis in biliary epithelia by aFas/Fas ligand-dependent mechanism. Am J Physiol. 1999 September; 277(3Pt 1): G599-608 (6). Ho L C, Singh M, Suresh G, Ng G C, Yap E H. Axenicculture of Blastocystis hominis in Iscove's modified Dulbecco's medium.Parasitol Res. 1993; 79(7): 614-6. (7). Crowell A L, Sanders-Lewis K A,Secor W E. In vitro metronidazole and tinidazole activities againstmetronidazole-resistant strains of Trichomonas vaginalis. AntimicrobAgents Chemother. 2003 April; 47(4): 1407-9.

Established methods are utilized to demonstrate additive and synergisticinteraction of DIM-related indoles and additional antiprotozoal agentsincluding methods established for P. Falciparum. (Gupta et al., 2002,Antimicrob Agents Chemother. 46(5):1510-5).

Examples of the agent(s) to be tested, both alone and in combination,for each species of protozoan parasite most appropriate for the methodsand compositions of the present invention are summarized in thefollowing five tables:

I. Plasmodium Species (P. Falciparum, P. Ovale, P. Vivex)

Class of Agent Example of Agent Concentration Range DIM-related indole3,3′-Diindolylmethane  .01-50 Micromolar Artimisinin derivativeArtesunate .001-10 Micromolar Curcumin or derivative Curcumin  .01-50Micromolar Choloriquine Chloroquine .001-20 Micromolar QuininePrimaquine .001-20 Micromolar Jasmonate Methyl jasmonate  .01-50Micromolar Antibiotic Clindamycin .001-20 Micromolar

II. Leishmania Species (L. donovani, L. tropica)

Class of Agent Example of Agent Concentration Range DIM-related indole3,3′-Diindolylmethane .01-50 Micromolar miltefosine miltefosine .01-50Micromolar Pentavalent Antimony Sodium stibogluconate .01-50 MicromolarAmphotericin B Amphotericin B .001-20 Micromolar  PentamidinePentamidine .001-20 Micromolar  Campothectins Topotecan .01-50Micromolar

III. Trypanosome Species (F. brucei, T. brucei gambiense, T. cruzi)

Class of Agent Example of Agent Concentration Range DIM-related indole3,3′-Diindolylmethane  .01-50 Micromolar Nifurtimox Nifurtimox .001-20Micromolar Benznidazole Benznidazole .001-20 Micromolar ArsenicalsMelarsoprol .001-20 Micromolar Campothectins topotecan .001-20Micromolar Ornithine decarboxylase eflornithine .001-20 MicromolarInhibitor

IV. Cryptosporidium Species (C. parvum, C. hominis)

Class of Agent Example of Agent Concentration Range DIM-related indole3,3′-Diindolylmethane .01-50 Micromolar Nitazoxanine Nitazoxanine.001-20 Micromolar  Curcumin Curcumin .01-50 Micromolar ArtemisininArtusenate .001-20 Micromolar  Flavonoid Genistein .01-50 Micromolar

V. Blastocystis Hominis

Class of Agent Example of Agent Concentration Range DIM-related indole3,3′-Diindolylmethane .01-50 Micromolar Curcumin Curcumin .01-50Micromolar Artemisinin Artusenate .001-20 Micromolar 

VI. Trichomonas Vaginalis

Class of Agent Example of Agent Concentration Range DIM-related indole3,3′-Diindolylmethane  .01-50 Micromolar Curcumin Curcumin  .01-50Micromolar Artemisinin Artusenate .001-20 Micromolar NitroimidazoleMetronidazole .001-20 Micromolar Nitroimidazole Tinidazole .001-20Micromolar

Expected results include the demonstration of antiprotozoal activity ofDIM-related indoles that is amplified when combined with selectedantiprotozoal agent(s).

5.10 Example 10 Use of DIM Alone and in Combination with Agents Using InVivo Models of Malaria

In malaria, sporozoites from the mosquito salivary glands rapidly enterthe circulation after a bite and localize in hepatocytes where theymultiply and develop into tissue schizonts. During this asymptomatictissue stage the sporozoites traverse through and damage multiplehepatocytes, yet the final infected hepatocyte has been shown to becomeresistant to apoptosis associated with the presence of the sporozoite.Anti-malarial activity against the primary liver stage is limited to afew of the known anti-protozoal drugs and has been limited to P.falciparum. The Atovaquone-Proguanil combination drug, Malarone, andPrimaquine show anti-liver stage activity but are expensive. Additionaltreatments active against liver stage infection are needed. Animalmodels using Plasmodium berghei sporozoites (Anka) in mice havedeveloped to test potential treatments for the liver stage followinginfection of the animals with sporozoites (van de Sand et al., 2005, MolMicrobiol. 58(3):731-42). Methods from this animal model are adapted todemonstrate the in vivo activity of DIM-related indoles against theliver stage of plasmodium infection. In addition this model lends itselfto demonstration of the induction of apoptosis in infected hepatocytesby DIM, the DIM-Artuscenate Combination, and the oral use ofDIM-Atuscenate-Curcumin, as well as other DIM-related indoleanti-protozoal combinations.

Briefly, the following method is used in mice to assay for sporozoiteinfectivity and hepatic infection in vivo. Female Swiss Webster mice, 5to 6 weeks old, are injected intravenously (i.v.) or intraperitoneally(i.p.) with DIM-related indole, alone or in combination with one or moreanti-protozoal agents, 60 min, 30 min, before i.v. injection of 10⁴ P.berghei sporozoites. Forty hours later, livers are harvested, total RNAis isolated, and malaria infection is quantified using reversetranscription followed by real-time PCR with primers that recognize P.yoelii-specific sequences within the 18S rRNA as previously described(Bruna-Romero et al., 2001, Int. J. Parasitol. 31:1499-1502). All invivo data are analyzed using the Student t test for unpaired samples.Typically, all experiments are performed twice with six mice per groupper experiment.

In vivo models have also been established to test for erythrocyticantimalarial activities of DIM-related indoles using the Plasmodiumvinckei petteri (279BY) strain and the Plasmodium yoelii nigeriensisstrain in female Swiss mice (Singh et al., 2000, Acta Trop.77(2):185-93). These methods are modified for intraperitoneal (i.p.) andoral administration of DIM-related indoles alone or in combination withother antimalarial agents. Drugs are injected i.p. or orally in 100 μlof DMSO. Parasitemia levels are monitored in Giemsa-stained bloodsmears, and blood samples are collected for determination of infectionon a fluorescence-activated cell sorter (Barkan et al., 2000, Int JParasitol. 30(5):649-53).

In testing in mice the following additional method is used to assay forefficacy of a DIM-related indole, alone or in combination with ananti-protozoal agent, against erythrocytic stages in vivo. The standard4-day suppression test is used to assess the efficacy of DIM and DIMcombinations against malaria erythrocytic stages in vivo (Peters, 1975,Ann. Trop. Med. Parasitol. 69:155-171). Female Swiss Webster mice, 5 to6 weeks old, are injected i.v. with 2×10⁵ GFP-expressing P. bergheiparasites, and 1 h later mice are injected i.v. with DIM, DIM plus asecond agent, or buffer for control group. Mice are treated with DIM,DIM plus a second agent, or buffer once daily for an additional 3 days.For the experiments in DIM or DIM in combination with a second agent,the test compounds are administered orally or parenterally, SwissWebster mice are infected with GFP-expressing parasites as above, and 1h later, DIM, or DIM plus a second agent, or water alone wasadministered by gavage. Following treatment, survival of the mice ismonitored and parasitemia is determined by fluorescence-activated cellsorting (FACS) analysis. For FACS, 2 μl of blood is diluted in 1 ml PBScontaining 1% fetal calf serum and 0.01% NaN₃, and the number offluorescent cells is determined for example using the FACS CaliburSystem with CellQuest Software (Becton Dickinson). Statisticalsignificance is determined using the Student t test for unpairedsamples. All experiments are performed twice with five mice per groupper experiment.

The treatment groups and dose ranges for in vivo testing of DIM-relatedindoles and complementary anti-protozoal agents in mice include thefollowing:

Antiprotozoal Agent Dose Range Route DIM 50-500 mg/kg/day Oral, i.p.*,i.v.** Artesunate 1-100 mg/kg/day Oral, i.p.*, i.v.** Artemether 1-100mg/kg/day Oral, i.p.*, i.v.** Dihydroartimisinin 1-100 mg/kg/day Oral,i.p.*, i.v.** Chloroquine 1-50 mg/kg/day Oral, i.p.*, i.v.** Atovoquone1-50 mg/kg/day Oral, i.p.*, i.v.** Quinine 1-50 mg/kg/day Oral, i.p.*,i.v.** Primaquine 1-50 mg/kg/day Oral, i.p.*, i.v.** Proguanil 1-50mg/kg/day Oral, i.p.*, i.v.** Curcumin 50-500 mg/kg/day Oral *i.p. =intraperitoneal; **i.v.= intravenous

5.11 Example 11 Use of the Combination of DIM, Artemesinin, and Cucurminin HIV-Associated Cryptosporidium Infection

Cryptosporidium sp. has been reported worldwide, especially infectingimmunocompromised persons and in immunocompetent persons having contactwith cattle. In individuals infected with human immunodeficiency virus(HIV), many will have cryptosporidiosis when diagnosed with HIVinfection and more will develop it later in the course of their disease.No specific effective treatment exists for cryptosporidiosis, and anintact immune system is the major factor to resolve the infection. Newapproaches for the treatment of cryptosporidiosis are needed, since manysymptomatic HIV infected patients do not have access to highly activeantiretroviral therapy (HAART). HIV infected patients with adequate CD4lymphocyte counts are reluctant to start HAART for the isolatedcomplaint of diarrhea. DIM-related indole therapy offers a newtherapeutic option for this common parasitic infection in both HIVpositive and negative individuals.

In this example of human use of oral DIM-based antiprotozoal treatment,an HIV positive male with chronic diarrhea will be treated with capsulescontaining absorption-enhanced DIM, Artemesinin, Curcumin, and piperine(Example 6.2). The subject is not on anti-retroviral therapy and his CD4lymphocyte count is greater than 200. He complains of chronic diarrheawith 3-4 unformed, watery stools daily for 1 month. Stool analysisreveals oocysts of cryptosporidium hominis/parvum. Following use of 2-4capsules twice daily of the DIM/Artemesinin/Cucumin mixture, improvementin diarrhea is expected. After 1 month of therapy it is expected thatsymptoms will resolve with resumption of a normal stool pattern and arepeat stool analysis will reveal no cryptosporidial oocysts. A similarapproach to treatment is used in HIV positive and negative individualsinfected with Cyclospora, Isospora belli, or Cystoisospora.

1. A method of treating or reducing the risk of a protozoal diseasecomprising administering to a subject in need thereof a therapeuticallyeffective amount of one or more DIM-related indoles and one or moreanti-protozoal agents.
 2. The method of claim 1, wherein said protozoaldisease is leishmaniasis or trypanosomiasis.
 3. The method of claim 1,wherein said protozoal disease is malaria, toxoplasmosis,cryptosporidiosis or bebesiosis.
 4. The method of claim 1, wherein saidprotozoal disease results from an infection from Microsporidia,Trichomonas, Cyclospora, Isospora or Blastocystis.
 5. The method ofclaim 1, wherein said protozoal disease results from an infection fromNeospora.
 6. The method of claim 2, 3 or 4, wherein said subject is ahuman.
 7. The method of claim 6, wherein said human isimmunocompromised.
 8. The method of claim 3, wherein said subject is alamb, calf, pig, rabbit or chicken.
 9. The method of claim 5, whereinsaid subject is a non-human mammal selected from the group consisting ofa dog, cow, sheep, goat and horse.
 10. The method of claim 1, whereinthe one or more DIM-related indoles are selected from the groupconsisting of: a compound of formula I:

wherein R³² and R³⁶ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, and ethoxycarbonylgroups, R³³ and R³⁷ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, R³¹, R³⁴, R³⁵, R³⁸,R⁴¹, and R⁴² are hydrogen, R⁵⁰, R⁵¹ are either hydrogen or methyl, andR⁹⁰, R⁹¹ are hydrogen; a compound of formula II:

wherein R⁶², R⁶³, R⁶⁶, R⁶⁷, R⁷⁰, and R⁷¹ are substituents independentlyselected from the group consisting of hydrogen, hydroxyl, and methoxy,and R⁶¹, R⁶⁴, R⁶⁵, R⁶⁸, R⁶⁹, R⁷², R⁸¹, R⁸² and R⁸³ are hydrogen; acompound of formula (III):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₆-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, and R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl, with the provisos that at leastone of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is otherthan hydrogen, and when R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are selectedfrom hydrogen, halo, alkyl and alkoxy, then R¹¹ and R¹² are other thanhydrogen and alkyl; a compound of formula (IV):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, with the proviso that one butnot both of R² and R⁶ is amino, mono-substituted amino, ordi-substituted amino; R¹¹ and R¹² are independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl,amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄alkyl, and di-(C₁-C₂₄ alkyl)amino-substituted C₁-C₂₄ alkyl, R¹³ and R¹⁴are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, with the provisothat at least one of R¹³ and R¹⁴ is other than hydrogen, and X is O, S,arylene, heteroarylene, CR¹⁵, R¹⁶ or NR¹⁷ wherein R¹⁵ and R¹⁶ arehydrogen, C₁-C₆ alkyl, or together form ═CR¹⁸, R¹⁹ where R¹⁸ and R¹⁹ arehydrogen or C₁-C₆ alkyl, and R¹⁷ is as defined for R¹¹ and R¹²; and acompound of formula (V):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², and X are defined asfor compounds of formula (III), and R²⁰ and R²¹ are defined as for R¹,R², R³, R⁴, R⁵, R⁶. R⁷ and R⁸.
 11. The method of claim 1, wherein theone or more DIM-related indoles are selected from the group consistingof diindolylmethane, hydroxylated DIMs, methoxylated DIMs,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR), hydroxylated LTRs,methoxylated LTRs, 5,5′-dimethylDIM (5-Me-DIM), 2,2′-dimethylDIM(2-Me-DIM), 5,5′-dichloroDIM (5-Cl-DIM),imidazolelyl-3,3′-diindolylmethane, nitro-substitutedimidazolelyl-3,3′-diindolylmethanes,2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole,6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole,2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane andindole-3-carbinol.
 12. The method of claim 11, wherein the DIM-relatedindole is DIM.
 13. The method of claim 12, wherein the DIM is processedDIM.
 14. The method of claim 1, wherein the DIM-related indole ismicroencapsulated with phosphatidylcholine (PC), complexed with PC, ormade into rapidly dissolving microparticles and nanoparticles.
 15. Themethod of claim 12, wherein the DIM is microencapsulated withphosphatidylcholine (PC), complexed with PC, or made into rapidlydissolving microparticles and nanoparticles.
 16. The method of claim 12,wherein the DIM is emulsified in a cream for topical administration tovaginal or rectal mucosa.
 17. The method of claim 1, wherein theanti-protozoal agent is selected from the group consisting ofatovaquone, amodiaquine, amphotericin, astemizole, butoconazole,clindamycin, eflornithine, fumagillin, iodoquinol, clioquinol,Etanidazole, Benznidazole, fluoroquinolones enoxacin, ciprofloxacin,doxycycline, melarsoprol, metronidazole, miltefosine, nifurtimox,nitazoxanide, paromomycin, pentamindine, sodium stibogluconate, antimonygluconate, suramin, suramin sodium salt, tinidazole, pyrimethamine,proguanil spiramycin, and sulfadoxine.
 18. The method of claim 1,wherein the anti-protozoal agent is selected from the group consistingof artemisinin, dihydroartemisinin, artemether, artesunate, quinine, aquinoline, halofantrine, mefloquine, lumefantrine, amodiaquine,pyronaridine, piperaquine, chloroquine, hydryoxychloroquine,napthoquine, primaquine, tafenoquine, amodiaquine, a 4-aminoquinoline, acurcuminoid, Genistein, dehydrosilybin, silybin A, silybin B, isosilybinA, isosilybin B, 8-(1; 1)-DMA-kaempferide, luteolin, baicalein,dihydrobetulinic acid, quercetin, eriodictyol acid, lursolic acid,oleanolic acid, and a triterpene.
 19. The method of claim 1, wherein theanti-protozoal agent is selected from the group consisting ofatovaquone, chloroquine, iodoquinol, clioquinol, Jasmonicacid[3-oxo-2-(2-pentenyl)cyclopentaneacetic acid], methyljasmonate[methyl 3-oxo-2-(2-pentenyl)cyclopentaneacetic acid],cis-jasmone[3-methyl-2-(2-pentenyl)-2-cyclopenten-1-one],3,3′-dihexyloxacarbocyanine iodide, sodium stibogluconate, curcumin,Pyrroloquinazolinediamine, Novobiocin, quercetin, cyclosporine,dihydrobetulinic acid, a campothecin, bortezimib, etoposide, salvicine,and an anthracycline.
 20. The method of claim 1, wherein theanti-protozoal agent is selected from the group consisting of an extractof Yucca schidigera, a tea or extract made from Artemisia annua, a teasor extract made from Curcuma domestica, an extract from garlic, a rootextract of Uvaria chamae or Hippocratea Africana, a root extract ofHomalium letestui, an extract of Vernonia brasiliana, an extract ofSapindus mukorossi, and an extract from Acanthospermum australe.
 21. Themethod of claim 1, wherein said therapeutically effective amount of oneor more DIM-related indoles and one or more anti-protozoal agents isadministered with a differentiation promoting agent.
 22. The method ofclaim 21, wherein said differentiation promoting agent is selected fromthe group consisting of vitamin D, a vitamin D derivative, calcitriol,vitamin A, a retinoid derivative, and a granulocyte/macrophage colonystimulating factor.
 23. The method of claim 1, wherein saidtherapeutically effective amount of one or more DIM-related indoles andone or more anti-protozoal agents is administered with an immunepotentiating agent.
 24. The method of claim 23, wherein said immunepotentiating agent is selected from the group consisting of an aloe veraextract, a mushroom extract, a beta-glucan, and an extract of the Panaxquinquefolium.
 25. The method of claim 1, wherein the one or moreDIM-related indoles and one or more anti-protozoal agents areadministered simultaneously.
 26. The method of claim 1, wherein the oneor more DIM-related indoles and one or more anti-protozoal agents areadministered within a short time of one another.
 27. The method of claim1, wherein the one or more DIM-related indoles are administeredintravenously, orally, topically, vaginally, or rectally.
 28. A methodof treating or reducing the risk of a protozoal disease comprisingadministering to a subject in need thereof a therapeutically effectiveamount of one or more DIM-related indoles.
 29. The method of claim 28,wherein said protozoal disease is leishmaniasis or trypanosomiasis. 30.The method of claim 28, wherein said protozoal disease is malaria,toxoplasmosis, cryptosporidiosis or bebesiosis.
 31. The method of claim28, wherein said protozoal disease results from an infection fromMicrosporidia, Trichomonas, Cyclospora, Isospora or Blastocystis. 32.The method of claim 28, wherein said protozoal disease results from aninfection from Neospora.
 33. The method of claim 29, 30 or 31, whereinsaid subject is a human.
 34. The method of claim 33, wherein said humanis immunocompromised.
 35. The method of claim 30, wherein said subjectis a lamb, calf, pig, rabbit or chicken.
 36. The method of claim 32,wherein said subject is a non-human mammal selected from the groupconsisting of dog, cow, sheep, goat and horse.
 37. The method of claim28, wherein the one or more DIM-related indoles are selected from thegroup consisting of: a compound of formula I:

wherein R³² and R³⁶ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, and ethoxycarbonylgroups, R³³ and R³⁷ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, R³¹, R³⁴, R³⁵, R³⁸,R⁴¹, and R⁴² are hydrogen, R⁵⁰, R⁵¹ are either hydrogen or methyl, andR⁹⁰, R⁹¹ are hydrogen; a compound of formula II:

wherein R⁶², R⁶³, R⁶⁶, R⁶⁷, R⁷⁰, and R⁷¹ are substituents independentlyselected from the group consisting of hydrogen, hydroxyl, and methoxy,and R⁶¹, R⁶⁴, R⁶⁵, R⁶⁸, R⁶⁹, R⁷², R⁸¹, R⁸², and R⁸³ are hydrogen; acompound of formula (III):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₆-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, and R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl, with the provisos that at leastone of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is otherthan hydrogen, and when R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are selectedfrom hydrogen, halo, alkyl and alkoxy, then R¹¹ and R¹² are other thanhydrogen and alkyl; a compound of formula (IV):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, with the proviso that one butnot both of R² and R⁶ is amino, mono-substituted amino, ordi-substituted amino; R₁₁ and R¹² are independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl,amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄alkyl, and di-(C₁-C₂₄ alkyl)amino-substituted C₁-C₂₄ alkyl, R¹³ and R¹⁴are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, with the provisothat at least one of R¹³ and R¹⁴ is other than hydrogen, and X is O, S,arylene, heteroarylene, CR¹⁵, R¹⁶ or NR¹⁷ wherein R¹⁵ and R¹⁶ arehydrogen, C₁-C₆ alkyl, or together form ═CR¹⁸, R¹⁹ where R¹⁸ and R¹⁹ arehydrogen or C₁-C₆ alkyl, and R¹⁷ is as defined for R¹¹ and R¹²; and acompound of formula (V):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², and X are defined asfor compounds of formula (III), and R²⁰ and R²¹ are defined as for R¹,R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸.
 38. The method of claim 28 where the oneor more DIM-related indoles are selected from the group consisting ofdiindolylmethane, hydroxylated DIMs, methoxylated DIMs,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR), hydroxylated LTRs,methoxylated LTRs, 5,5′-dimethylDIM (5-Me-DIM), 2,2′-dimethylDIM(2-Me-DIM), 5,5′-dichloroDIM (5-Cl-DIM),imidazolelyl-3,3′-diindolylmethane, nitro-substitutedimidazolelyl-3,3′-diindolylmethanes,2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole,6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole,2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane andindole-3-carbinol.
 39. The method of claim 38, wherein the DIM-relatedindole is DIM.
 40. The method of claim 39, wherein the DIM is processedDIM.
 41. The method of claim 28, wherein the DIM-related indole ismicroencapsulated with phosphatidylcholine (PC), complexed with PC, ormade into rapidly dissolving microparticles and nanoparticles.
 42. Themethod of claim 39, wherein the DIM is microencapsulated withphosphatidylcholine (PC), complexed with PC, or made into rapidlydissolving microparticles and nanoparticles.
 43. The method of claim 39,wherein the DIM is emulsified in a cream for topical administration tovaginal or rectal mucosa.
 44. The method of claim 28, wherein saidtherapeutically effective amount of one or more DIM-related indoles isadministered with a differentiation promoting agent.
 45. The method ofclaim 44, wherein said differentiation promoting agent is selected fromthe group consisting of vitamin D, a vitamin D derivative, calcitriol,vitamin A, a retinoid derivative, and a granulocyte/macrophage colonystimulating factor.
 46. The method of claim 28, wherein saidtherapeutically effective amount of one or more DIM-related indoles isadministered with an immune potentiating agent.
 47. The method of claim46, wherein said immune potentiating agent is selected from the groupconsisting of an aloe vera extract, a mushroom extract, a beta-glucan,and an extract of the Panax quinquefolium.
 48. The method of claim 28,wherein the one or more DIM-related indoles are administeredintravenously, orally, topically, vaginally, or rectally.
 49. Acomposition comprising a therapeutically effective amount of thecombination of one or more DIM-related indoles and one or moreanti-protozoal agents.
 50. The composition of claim 49, wherein the oneor more DIM-related indoles are selected from the group consisting of: acompound of formula I:

wherein R³² and R³⁶ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, and ethoxycarbonylgroups, R³³ and R³⁷ are substituents independently selected from thegroup consisting of hydrogen, hydroxyl, and methoxy, R³¹, R³⁴, R³⁵, R³⁸,R⁴¹, and R⁴² are hydrogen, R⁵⁰, R⁵¹ are either hydrogen or methyl, andR⁹⁰, R⁹¹ are hydrogen; a compound of formula II:

wherein R⁶², R⁶³, R⁶⁶, R⁶⁷, R⁷⁰, and R⁷¹ are substituents independentlyselected from the group consisting of hydrogen, hydroxyl, and methoxy,and R⁶¹, R⁶⁴, R⁶⁵, R⁶⁸, R⁶⁹, R⁷², R⁸¹, R⁸², and R⁸³ are hydrogen; acompound of formula (III):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₆-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, and R¹¹ and R¹² areindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkoxycarbonyl, amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄alkylamino)-substituted C₁-C₂₄ alkyl, and di-(C₁-C₂₄alkyl)amino-substituted C₁-C₂₄ alkyl, with the provisos that at leastone of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² is otherthan hydrogen, and when R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are selectedfrom hydrogen, halo, alkyl and alkoxy, then R¹¹ and R¹² are other thanhydrogen and alkyl; a compound of formula (IV):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are substituentsindependently selected from the group consisting of hydrogen, C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl,C₆-C₂₄ aralkyl, halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, halocarbonyl, C₂-C₂₄alkylcarbonato, C₆-C₂₀ arylcarbonato, carboxy, carboxylato, carbamoyl,mono-(C₁-C₂₄ alkyl)-substituted carbamoyl, di-(C₁-C₂₄ alkyl)-substitutedcarbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl, carbamido,cyano, isocyano, cyanato, isocyanato, isothiocyanato, azido, formyl,thioformyl, amino, mono- and di-(C₁-C₂₄ alkyl)-substituted amino, mono-and di-(C₅-C₂₀ aryl)-substituted amino, C₂-C₂₄ alkylamido, C₅-C₂₀arylamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,phosphono, phosphonato, phosphinato, phospho, phosphino, andcombinations thereof, and further wherein any two adjacent (ortho)substituents may be linked to form a cyclic structure selected fromfive-membered rings, six-membered rings, and fused five-membered and/orsix-membered rings, wherein the cyclic structure is aromatic, alicyclic,heteroaromatic, or heteroalicyclic, and has zero to 4 non-hydrogensubstituents and zero to 3 heteroatoms, with the proviso that one butnot both of R² and R⁶ is amino, mono-substituted amino, ordi-substituted amino; R¹¹ and R¹² are independently selected from thegroup consisting of hydrogen, C₁-C₂₄ alkyl, C₂-C₂₄ alkoxycarbonyl,amino-substituted C₁-C₂₄ alkyl, (C₁-C₂₄ alkylamino)-substituted C₁-C₂₄alkyl, and di-(C₁-C₂₄ alkyl)amino-substituted C₁-C₂₄ alkyl, R¹³ and R¹⁴are defined as for R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, with the provisothat at least one of R¹³ and R¹⁴ is other than hydrogen, and X is O, S,arylene, heteroarylene, CR¹⁵, R¹⁶ or NR¹⁷ wherein R¹⁵ and R¹⁶ arehydrogen, C₁-C₆ alkyl, or together form ═CR¹⁸, R¹⁹ where R¹⁸ and R¹⁹ arehydrogen or C₁-C₆ alkyl, and R¹⁷ is as defined for R¹¹ and R¹²; and acompound of formula (V):

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², and X are defined asfor compounds of formula (III), and R²⁰ and R²¹ are defined as for R¹,R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸.
 51. The composition of claim 49, whereinthe one or more DIM-related indoles are selected from the groupconsisting of diindolylmethane, hydroxylated DIMs, methoxylated DIMs,2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTR), hydroxylated LTRs,methoxylated LTRs, 5,5′-dimethylDIM (5-Me-DIM), 2,2′-dimethylDIM(2-Me-DIM), 5,5′-dichloroDIM (5-Cl-DIM),imidazolelyl-3,3′-diindolylmethane, nitro-substitutedimidazolelyl-3,3′-diindolylmethanes,2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo-[2,3-b]carbazole,6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole and2,10-dicarbethoxy-6-ethoxycarbonyloxy-5,7-dihydro-indolo-[2,3-b]carbazole,and 2,6-dicarbethoxy-3,3′-dimethyl-13,14-diindolylmethane.
 52. Thecomposition of claim 49, wherein the anti-protozoal agent is selectedfrom the group consisting of atovaquone, amodiaquine, amphotericin,astemizole, butoconazole, clindamycin, eflornithine, fumagillin,iodoquinol, clioquinol, Etanidazole, Benznidazole, fluoroquinolones,enoxacin, ciprofloxacin, doxycycline, melarsoprol, metronidazole,miltefosine, nifurtimox, nitazoxanide, paromomycin, pentamindine, sodiumstibogluconate, antimony gluconate, suramin, suramin sodium salt,tinidazole, pyrimethamine, proguanil spiramycin, and sulfadoxine. 53.The composition of claim 49, wherein the anti-protozoal agent isselected from the group consisting of artemisinin, dihydroartemisinin,artemether, artesunate, quinine, a quinoline, halofantrine, mefloquine,lumefantrine, amodiaquine, pyronaridine, piperaquine, chloroquine,hydryoxychloroquine, napthoquine, primaquine, tafenoquine, amodiaquine,a 4-aminoquinoline, a curcuminoid, Genistein, dehydrosilybin, silybin A,silybin B, isosilybin A, isosilybin B, 8-(1; 1)-DMA-kaempferide,luteolin, baicalein, dihydrobetulinic acid, quercetin, eriodictyol acid,lursolic acid, oleanolic acid, and a triterpene.
 54. The composition ofclaim 49, wherein the anti-protozoal agent is selected from the groupconsisting of atovaquone, chloroquine, iodoquinol, clioquinol, Jasmonicacid [3-oxo-2-(2-pentenyl)cyclopentaneacetic acid], methyljasmonate[methyl 3-oxo-2-(2-pentenyl)cyclopentaneacetic acid],cis-jasmone[3-methyl-2-(2-pentenyl)-2-cyclopenten-1-one],3,3′-dihexyloxacarbocyanine iodide, sodium stibogluconate, curcumin,Pyrroloquinazolinediamine, Novobiocin, quercetin, cyclosporine,dihydrobetulinic acid, a campothecin, bortezimib, etoposide, salvicine,and an anthracycline.
 55. The composition of claim 49, wherein theanti-protozoal agent is selected from the group consisting of an extractof Yucca schidigera, a tea or extract made from Artemisia annua, a teasor extract made from Curcuma domestica, an extract from garlic, a rootextract of Uvaria chamae or Hippocratea Africana, a root extract ofHomalium letestui, an extract of Vernonia brasiliana, an extract ofSapindus mukorossi, and an extract from Acanthospermum australe.
 56. Thecomposition of claim 49, wherein the DIM-related indole is DIM.
 57. Thecomposition of claim 56, wherein the DIM is processed DIM.
 58. Thecomposition of claim 49, wherein the DIM-related indole ismicroencapsulated with phosphatidylcholine (PC), complexed with PC, ormade into rapidly dissolving microparticles and nanoparticles.
 59. Thecomposition of claim 56, wherein the DIM is microencapsulated withphosphatidylcholine (PC), complexed with PC, or made into rapidlydissolving microparticles and nanoparticles.
 60. The composition ofclaim 49, wherein the composition is formulated for oral administration,intravenous administration, vaginal administration, rectaladministration or topical administration.
 61. The composition of claim49 or 57, wherein the composition is formulated as a dietary supplement,fortified food, drink mix, or tea mix.
 62. The composition of claim 49,further comprising Vitamin-E polyethyleneglycol succinate (TPGS),piperine, limonine, D-Limonene or polyethyleneglycol.
 63. Thecomposition of claim 56 or 57, wherein the composition is formulated fororal administration.
 64. The composition of claim 56, wherein the DIMand the antiprotozoal agent are emulsified in a cream for topicaladministration to vaginal or rectal mucosa.